Antimicrobial activity of agnihotra ash against common pathogens

Antimicrobial activity of Agnihotra
ash against common pathogens
Dr. Bhoj R Singh*, Dr. U Berk, Dr. AB Pande, Dr.
DK Sinha, Dr. R. Karthikeyan, Dr. Akanksha Yadav
Email: br.singh@icar.gov.in
वेदों क
े अनुसार यज्ञ पााँच प्रकार क
े हैं – ब्रह्मयज्ञ, देवयज्ञ, पितृयज्ञ, वैश्वदेवयज्ञ और
अततथियज्ञ.
देवयज्ञ, वेदी बनाकर, अग्नन प्रज्जज्जवलित कर जो होम ककया जाता है वही अग्ननहोत्र यज्ञ है.
अग्ननहोत्र यज्ञ का वर्णन ‘यजुवेद’ में है. अग्ननहोत्र एक ऐसा होम (आहुतत) है ग्जसे
प्रततददन ककया जाता है तथा उसकी अग्नन को बुझने नहीीं ददया जाता.
सूयोदय और सूयाणस्त क
े समय ही यह यज्ञ करने का महत्त्व है.
सूयोदय क
े समय तिम्िलिखित मन्त्र का उच्चारण करिा चाहिए –
सूयाणय स्वाहा सूयाणय इदम ् न मम .
प्रजापतये स्वाहा प्रजापतये इदम् न मम ..
सूयाास्त क
े समय तिम्िलिखित मन्त्र का उच्चारण करिा चाहिए –
अननये स्वाहा अननये इदम् न मम .
प्रजापतये स्वाहा प्रजापतये इदम् न मम ..’

Agnihotra and infections
• Agnihotra fire performed at specified timing (sunrise and sunset) in a copper
pyramid while chanting Vedic Mantra.
• Agnihotra is said to purify the environment and especially and specially shown
to mitigate water pollution problem. (Berk and Sharma, 2015).
• Preliminary tests have shown that if Agnihotra ash added to polluted water,
water gets purified (Mondkar, 1982; Gerlecka, 1988; Matlander; 2013;
Sharma, 2011).
• It is not only ash of the ingredients used in Agnihotra but the Pran infused due
to Vedic Mantra in Agnihota potentiate the purifying activity of the Agnihotra
ashes (Sharma, 2011).
• It is postulated that electromagnetic spectrum is generated under the influence
of solar energy and sonic potential of Mantra, the numinous sound which plays
pivotal role in purifying effect of Mantra (Rathod , 2020) and recommended
Agnihotra to be performed at homes, hospitals, public places at regular basis
even to combat epidemics.
• Rigveda, clarifies that smoke from Agnihotra yagnya spreads in space or
surrounding and directs arrow towards the viruses and bacteria make them to
vanish (Concept of Sacrifice Vaina, 2020, volume 87 of 10. Rig veda).
• Most of the literature specifies burning of herbs known to posses several
antimicrobial active ingredients in Agnihotra Yagnya and it is often countered
by scientists that it is the fumes of herbs that kills microbes and mantra not
have any significant role to play but observations of Berk and Coworkers
counter it and proclaim central role of Vedic mantras instead of Hawan
Samagri.
23-02-2021 2
Division of Epidemiology, ICAR-IVRI,
Izatnagar, India

Hypothesis
Vedic Mantra have/ don’t have pivotal role in conferring
antimicrobial effect to Agnihotra Ash
• For proving we need ashes from Agnihotra performed
methodically with and without chanting Vedic mantras and
also the ashes of cow, and buffalo dung cakes burnt at
farmers’ homes.
• The standard potentially pathogenic microbial strains
found in contaminated food and water.
• Experimentation on survival of bacteria in sterilized tap
water with and without adding ash(es) using standard
microbiologically accepted protocol.
23-02-2021 3
Izatnagar, India

Sources of Mantra-yukt (with Vedic mantra) and
Mantra-viheen (without Vedic mantra)
Agnihotra Ash and Control Ash
1. Dr. Ulrich Berk, President, German Association of Homa Therapy, Germany.
2. Dr. AB Pande, Bareilly, UP, India
3. Fivefold Path, Ladvi, Maheshwar, MP, India
4. Cow (Shahiwal) dung cake Ash, Farmer, Bareilly, UP.
5. Buffalo (Murrah) dung cake Ash, Farmer, Bareilly, UP.
6. Mix Cow (Shahiwal) and Buffalo (Murrah) dung cake Ash, Farmer, Bareilly,
UP
From Farmers: Dung cake ashes were collected in the morning from Barosi, a
special fireplace to burn dung cakes of the farmers in nearby village
All ashes were collected in only paper packs/ bags, no plastic/ polythene bags were
used.
Weighing was done aseptically and desired quantities (1 g) were made in to िुड़िया,
specially wrapped in sterilized paper. All ash wraps were kept at room
temperature in steel box till used.
23-02-2021 4
Izatnagar, India

Microbes used
Microbial strains available as stock in the Division of Epidemiology were revived and
confirmed as per standard protocol (Carter, 1975; Singh, 2009).
Strain No. Identity Strain No. Identity
FKidHLY Aeromonas popoffii RadhikaLWSNH2 Klebsiella pneumoniae ssp.
pneumoniae
10WErt Alacligenes faecalis 2201DAbBM Klebsiella pneumoniae ssp.
pneumoniae
921BVSNH2C Bacillus megaterium Movis Moraxella ovis
CAb4 Candida albicans RadhikaLBPColR Proteus mirabilis
CAb5 Candida albicans 404THP1 Pseudomonas aeruginosa
KPUMR3 Candida tropicalis biovar
urealyticus
192TCHBNHF Salmonella enterica ssp. enterica ser
Naestved
185WTUHly Enterococcus faecium ATCC29312 Staphylococcus aureus
1337DWSNH2 Enterococcus malodoratus ATCC43300 Staphylococcus aureus
VTSWL Erwinia cacticida 972CPSNHL Staphylococcus capitis ssp. capitis
581CMFRα Erwinia stewartii 25 DFSHLY Staphylococcus caseolyticus
2201DAbAM Escherichia coli 25DEST Staphylococcus chromogenes
LB1L Escherichia coli NDM 2201DAbBHly Staphylococcus haemolyticus
2799DFSW Escherichia fergusonii 18TSP Staphylococcus hominis
VTSWS Hafnia alvei
23-02-2021 5
Izatnagar, India

Methods
Ash extracts: Aqueous extract from different ashes (Agnihotra and Control) was made as
described in Agnihotra Manual (Berk, 2017).
• Agnihotra ashes (with and without mantra) from AB Pandey, Dr. Ulrich Berk and from
Maheshwar used to make the extracts. Cow dung cake ash extract as Control Ash.
Ash extract in Water(AAW): 5 g Ash was mixed in 1 L water boiled for 1 h and then boiled
for 10 more min, kept overnight, filtered to make it bacteria free and tested for
antimicrobial activity.
Ash extract in DMSO (AAD): One g of ash was mixed in 10 mL DMSO, shaken for 15 min
and then allowed to stand overnight and supernatant taken to test antimicrobial activity.
Ash extract in methanol (AAM): 1 gm Ash mixed in 10 mL Methanol, shaken for 15 min
and then allowed to stand overnight and supernatant taken to test for antimicrobial
activity.
A total of 27 microbial strains were tested using
– Agar well diffusion assay (50 µL extract filled in the well) for each microbial strain
in triplicate (Singh, 2013).
– Micro-dilution method (200 µL in each well) in LB broth using 96 well plate starting
from 80% DMSO and aqueous extracts and 50% of methanolic extract to 10%, in
triplicate and wells were inoculated with 1.5 µL the test culture (in growing phase at
0.2 OD540) in triplicate. Incubated at required temperature and time and then read
for apparent growth.
23-02-2021 6
Izatnagar, India

Testing antibacterial Activity of Ashes
mixed in sterilized tap water
• Four bacteria were used for the purpose:
1) Alacligenes faecalis 2) Escherichia coli NDM 3) Pseudomonas aeruginosa
and 4) Staphylococcus aureus
• Tap-water, 100 mL in each screw capped bottles (MD bottles) was sterilized at
121oC for 30 min and allowed to cool down at room temperature.
• To test each bacteria 100 µL the test culture (in growing phase at 0.2 OD540)
was inoculated in triplicate for each ash and one bottle was inoculated as
control without any ash.
• Each ash was mixed individually and aseptically in culture inoculated bottles,
shaken for 10 min and allowed to stand undisturbed for two hours and then
counting of colony forming units was done after making decimal dilutions
from aseptically drawn 100 µL of water using Miles and Misra method (1938)
in triplicate for each bottle at day 0, day one (after 24 h) and day two (after 48
h).
• Decimal Microdilution from each bottle were also made in triplicate in 96 well
tissue culture plate, each well containing 180 µL of sterilized distilled water
and dilutions were made by successively transferring 20 µL of test water. From
the MD bottles already inoculated. Thereafter, 60 µL of sterilized LB broth was
added into each well aseptically. Plates were incubated at 37oC for 24 hr and
then growth was noted in the wells. The last dilution well showing the growth
was considered as the Log10 count of the bacteria in the test bottle. Count was
done on day 0, 1, 2, 3, 4, 7, 10, 14, 35 after inoculation and adding the ash.
23-02-2021 7
Izatnagar, India

Results of antimicrobial activity of
Ash extracts
• None of 27 microbes tested was visibly (growth inhibition zone in agar
well diffusion assay and turbidity in LB broth in 96 well culture plate )
inhibited either by control or Agnihotra ashes by any of the three kinds
of extracts i.e., AAW, AAD and AAM.
• The study indicated that nothing with antimicrobial potential can be
extracted out of ashes (Agnihotra and Control) using the three
different extractions methods used in the study.
• Observations are in contradiction to earlier reports of antimicrobial
potential of Agnihotra ash. Reasons may be many as type of bacteria,
strains of bacteria, methodology variations etc.
23-02-2021 8
Izatnagar, India

Results of antibacterial activity of Ashes mixed in
sterilized tap water
6.29
5.04
8.96
6.35
5.46
4.48
5.31
6.19 6.31
5.07
5.51
5.31
7.54
8.29
6.19
5.31
3.31
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
0 h 24h 48h
Log10
of
colonyforming
units
of
bacteria
Hours after inoculation
Survival of Escherichia coli in presence of different ashes
mixed (1% w/v) in sterilized tap water
No Ash Control UB ash
Agnihotra UB Ash Control AB Ash
Agnihotra AB Ash Control M Ash
Agnihotra M Ash Cow dung cake Ash
Buffalo dung cake Ash Mix dung cake Ash
23-02-2021 9
Izatnagar, India

5.40
6.23
6.63
5.30
4.36 4.28
4.88
1.98
5.51
4.31 4.33
11.43
5.47 5.79
11.03
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
0 h 24h 48h
Log10
of
colonyforming
units
of
bacteria
Survival of Staphylococcus aureus in presence of different
ashes mixed (1% w/v) in sterilized tap in water
23-02-2021 10
Izatnagar, India

5.49
4.69
5.31
2.31
5.31
6.55
3.31
5.57
5.70
5.37
5.24
6.18
4.43
2.82
1.11
5.47
3.15
2.39
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
0 h 24h 48h
Log10
of
colonyforming
units
of
bacteria
Survival of Alcaligenes faecalis in presence of different
23-02-2021 11
Izatnagar, India

5.95
7.87
9.03
5.69
5.05
3.84
5.65
4.77
2.97
5.50
11.72 11.79
5.45
4.95
6.74
4.63
3.62 3.75
5.79
4.55
7.06
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
0 h 24h 48h
Log10
of
colonyforming
units
of
bacteria
Survival of Paseudomonas aeruginosa in presence of different
23-02-2021 12
Izatnagar, India

Summary of the Experiment for short term (24-48 h)
survival of bacteria in presence of different ashes
Added in contaminated water
at 1% W/V
Escherichia
coli
Staphylococcus
aureus
Alkaligenes
faecalis
Pseudomonas
aeruginosa
No Ash 1 1 1 1
Control UB ash 1 1 1 1
Agnihotra UB Ash 1 2 1 1
Control AB Ash 1 1 1 2
Agnihotra AB Ash 2 2* 1 2
Control M Ash 1 1 1 1
Agnihotra M Ash 1 1 1 1
Cow dung cake Ash 1 1 1 2*
Buffalo dung cake Ash 1 1 2 2
Mix dung cake Ash 2 2* 2 2*
* decreased (p, 001, ANOVA) when counted after 24 h but increased on counting at 48 h of
adding the ash
23-02-2021 13
Izatnagar, India

Observations on Experiment for long term
survival of bacteria in presence of different ashes
5
12 12 12 12 12 12 12 12
5 5
12 12 12 12 12
8
5
6 6
7
8
7
6
3
4
5
6
5 5
6
10
8
1
6
5
5
7
8
12 12 12
8
4 4
5
4
3
5
12 12 12
8
5
0
2
4
6
8
10
12
14
0 1 2 3 4 7 10 14 35
Growth
upto
Log10
(Decinal)
dilution
Days after inoculation
Long term survival of Escherichia coli in presence of different
ashes mixed (1% w/v) in sterilized tap water
Agnihotra AB Ash Buffalo dung cake ash
Cow dung cake ash Mix dung cake Ash
23-02-2021 14
Izatnagar, India

5
8
12 12 12 12 12 12 12
5
4 4
8
9
12
5
8
6
6
7
9 9
8
7 7 7
5
6
3
5
8
10
8
7 7
5
5
6
7
9
12 12
11
7
8
5 5
6 6
12 12
5
3
2
5
4
5 5
12 12
5
3
2
0
2
4
6
8
10
12
14
0 1 2 3 4 7 10 14 35
Growth
upto
Log10
(Decinal)
dilution
Long term survival of Staphylococcus aureus in presence of different
ashes mixed (1% w/v) in sterilized tap water
No Ash Control UB ash Agnihotra UB Ash
Control AB Ash Agnihotra AB Ash Buffalo dung cake ash
23-02-2021 15
Izatnagar, India

3
8
12 12 12 12 12
8
5
5 5
7
6
5
4
3
4
5
3
1 1 1
12 12
7
4
3
3
4 4
10
12 12 12
8
5
0
2
4
6
8
10
12
14
0 1 2 3 4 7 10 14 35
Growth
upto
Log10
(Decinal)
dilution
Long term survival of Alcaligenes faecalis in presence of
different ashes mixed (1% w/v) in sterilized tap water
No Ash
Control UB ash
Agnihotra UB Ash
Control AB Ash
Agnihotra AB Ash
Buffalo dung
cake ash
Cow dung cake
ash
Mix dung cake
Ash
23-02-2021 16
Izatnagar, India

6
12 12 12 12 12 12
16 16
12
6
5
4
2
7
8
9
12 12
6
5
4 4
12 12
7
4 4
6
5
6 6
12 12
10
7
6
6
5
8
10
12 12 12
8
6
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 7 10 14 35
Growth
upto
Log10
(Decinal)
dilution
Long term survival of Pseudomonas aeruginosa in presence of
different ashes mixed (1% w/v) in sterilized tap water
Agnihotra AB Ash Buffalo dung cake ash
23-02-2021 17
Izatnagar, India

Conclusions
• None of Agnihotra ashes (with or without mantra) and dung cake ash of cow
and buffalo was as good antimicrobial as ash from mixed (cow and buffalo)
dung cake ash. Mixed dung cake ash significantly (p, 0.001) reduced count of
all four types of bacteria (A. faecalis, E. coli, P. aeruginosa and S. aureus).
• Buffalo dung cake ash was superior to cow dung cake ash in significantly
reducing count of A. faecalis and P. aeruginosa, however, both had no effect on
E. coli and S. aureus survival.
• Agnihotra ash (with mantras) from Dr. AB Pandey significantly reduced the
load of (E. coli, P. aeruginosa and S. aureus) in contaminated water. However,
Agnihotra ash (without mantras) from Dr. AB Pandey also reduced significantly (as
good as ash from agnihotra with mantras) count of P. aeruginosa but not of E. coli
and S. aureus indicating effect of mantra.
• Neither of the Agnihotra ashes from Maheshwar and without mantra ash from
Dr. U Berk had any significant antimicrobial effect on survival of any of the four
bacteria tested but ash from Agnihotra with mantra from Dr. U Berk
significantly reduced count of S. aureus within 24 h without any significant
effect on other three bacteria. Indicating effect of mantra.
23-02-2021 18
Izatnagar, India

• The study indicated that variation in antimicrobial activity of
Agnihotra ashes might be the due to variability in effect of
different mantra or difference in the Hawan Samgri ingredients
used in Agnihotra, Dr. Pandey used regular Hawan Samagri and
Gayatri mantra while Dr. Berk and at Maheshwar Agnihotra was
performed using cow dung cake , cow ghee and rice with Surya
and Prajapti mantra.
• Extract of Agnihotra ashes and other ashes shown no
antimicrobial potential irrespective of the extraction procedure
and solvent used i.e, antimicrobial potential of ashes are
inextractable with used methods.
• Different mantras may confer different spectrum of
antimicrobial activity to the Agnihotra ashes.
23-02-2021 19
Izatnagar, India

Long way to go!!!
The study revealed that, though mantra in Agnihotra conferred antimicrobial potential to
ashes produced but differed with different mantra and different ingredients of materials
burnt in Agnihotra. Thus these is need for
– High precision analysis of ashes from Agnihotra with and without mantra.
– Evaluation of antimicrobial activity of Agnihotra ashes generated with use of
different mantras (Gayatri, Surya, Prajapati, Mahamrityunjay, Rudra Gayatri and
Surya Gayatri, Prakhar Pragya, Sajal Shraddha, Shanti mantras etc.) using the
same Hawan Samagri and same procedure against the same strains of different
pathogens.
– Determination of spectrum of antimicrobial activity of different mantra used in
Agnihotra singly and in combination.
– Determination and differentiation of cidal (lethal) and static effect of different
Agnihotra ashes and mantra.
– Determination of antimicrobial spectrum of each and every or at least the most
important Agnihotra mantras (Gayatri, Surya, Prajapati, Mahamrityunjay, Rudra
Gayatri and Surya Gayatri, Prakhar Pragya, Sajal Shraddha, Shanti mantras etc.)
without burning any Hawan Samagri.
– Instead to sticking only to cow products used in Homa, other domestic animals like
buffalo, goat, sheep, camel etc. products needs to be evaluated. In the present study
ash from mixed dung cakes yielded the best antimicrobial results thus more
elaborate studies are essential to think of therapeutic use of Agnihotra ashes.
23-02-2021 20
Izatnagar, India

अग्ननहोत्र यज्ञ से उत्पन्न अग्नन से ‘रज और तम’ कर्ों का नाश होता है-यजुवेद
Thanks for your patience
23-02-2021 21
Izatnagar, India

Antimicrobial activity of agnihotra ash against common pathogens

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