This study aimed to isolate and characterize bacteria and fungi from homemade cigarettes and bidis. Samples were collected and plated using serial dilution techniques. Isolates were identified through microscopic examination, biochemical tests, and comparing characteristics to literature. Four fungal isolates - Aspergillus, Mucor, Saccharomyces, and Penicillium - were identified from bidis. Three fungi - Aspergillus, Saccharomyces, and Penicillium - were found in homemade cigarettes. Two bacterial isolates, Staphylococcus aureus and Streptococcus pyogenes, were identified from bidis. The presence of pathogens in tobacco smoke may explain high rates of respiratory illness in smokers. Increased awareness of microbial content could

1. Research augmented by: Plabita Dolakasaria , M. Sc. Research
Research compiled by: Dr. Avra Pratim Chowdhury
M. Phill., M. Sc., Ph.D. Research Scholar, Gauhati University.
Place of study:
The project work was carried out in the Microbiology Laboratory,
Department of Microbiology,
College of Allied Health Sciences,
Assam down town University, Guahati.

2. CONTENTS
BACKGROUND
HISTORY
The purpose of the study is
to put a special emphasis
on presence of micro
organisms in bidi and
home made cigarette
sample as collected.
AIM AND
OBJECTIVES
METHODOLOGY
ISOLATION and
IDENTIFICATION
SIGNIFICANCESCREENING OF
ISOLATES
CONCLUSION REFERENCE

3. Biddi is a thin cigarette or mini-
cigar filled with tobacco flake and
commonly wrapped in a Diospyros
melanoxylonor or Piliostigma
racemosum leaf tied with a string or
adhesive at one end.
 Homemade cigarette sample used
for this study was made by chopping
of tobacco leaf and rolling it with a
straw paper. The prevalence of bidi
and home made cigarette smoking is
seen to be more among people of
rural areas with very little
information available about
concentration of toxic agents
present in them.
 Scientist Paul Verweij,
the University Medical
Centre in Nijmegen,
Netherlands stated that
“ Tobacco is a source of
fungal spores”.
International Association for Research
on Cancer (IARC) stated that tobacco
smoke contains endotoxin (lipo
polysaccharide, LPS), a family of
inflammatory toxins from Gram-
negative bacteria known to cause
respiratory disease upon inhalation.

4. AIM: The present study is aimed at
Isolation and characterization of bacteria
and fungi from home made cigarette and bidi
OBJECTIVES:
Isolation of bacteria and fungi from bidi and home made cigarette
sample.
Identification and characterization of bacterial and fungal isolates
(microscopic study, macroscopic study, biochemical tests.)
Assessment of role of the microbial isolates in tobacco related
disease.
To create awareness among people about the effects of pathogenic
microorganisms in the above samples.
Duration of study: The study was conducted from January 2018 to April 2018.

5.  COLLECTION OF
SAMPLE:
Bidi sample was
purchased from local
shop of North
Lakhimpur district
of Assam and home
made cigarette
sample was collected
from a local person
of a village of North
Lakhimpur. The
sample were carried
to the laboratory in a
separated zip-lock
pouch
ISOLATION OF BACTERIA AND
FUNGI:
Isolation of the bacteria and
fungi was done by following serial
dilution and later growing on various
media by pour plate method.
METHODOLOGY
CULTURAL
CHARACTERISTICS:
The colony morphology of
the bacteria and fungi was determined
by observing the size, form, color,
edge, elevation, margin, texture and
opacity of the colony in the media.

6.  MICROSCOPIC STUDY:
I. GRAM STAINING- Microscopic study of bacteria was done by gram
staining method
II. LACTO PHENOL COTTON BLUE (LPCB) STAINING- Microscopic
study of fungal colony was done by Lacto phenol cotton blue staining
method.
 BIOCHEMICAL CHARACTERIZATION:
Biochemical test for identification of bacterial isolates include-
1. Coagulase Test
2. Catalase Test
3. MR Test
4. VP Test
5. Indole Test
6. Citrate Test

7. Table : Cultural characteristics of bacterial isolates obtained from bidi
Fig : Bacterial colonies isolated from bidi
Table : Microscopic characteristic of bacterial isolates obtained from bidi

8. Table 1: Cultural characteristics of fungal isolates obtained from bidi
Fig: Fungal colonies isolated from Bidi
Plate No Form Color Elevation Margin Appearance Opacity Texture No of
Colony
1(Bidi) Circular Black center
with white
border
Raised Entire Dull Opaque Velvety 4
1(Bidi) Irregular Cream Raised Entire Shiny Opaque Smooth 2
2(Bidi) Circular Black to
brown
Raised Entire Dull Opaque Smooth 3
2(Bidi) Circular Bluish green
with white
border
Raised Entire Cottony Opaque Smooth 3
3(Bidi) Circular Dirty white
with yellow
center
raised Entire Cottony Opaque Fluffy 2
3(Bidi) Circular Black with
White
Border
Raised Entire Dull Opaque Velvety 1
Fig: Bidi

9. Plate
No
Form Color Elevation Margin Appearance Opacity Texture No of
Colony
1(H) Circular Black with
white border
Raised Entire Dull Opaque Velvety 4
1(H) Circular Bluish green
with white
border
Raised Entire Dull Opaque Velvety 2
1(H) Irregular Cream Slightly
raised
Entire Shiny Opaque smooth 2
2(H) Circular Cream Slightly
raised
Entire Dull Opaque Smooth 1
2(H) Circular Black with
White Border
Raised Entire Dull Opaque Velvety 4
3(H) Circular Greenish Raised Entire Dull Opaque Velvety 3
3(H) Circular Black center
with white
border
Raised Entire Dull Opaque Velvety 4
Table 2: Cultural characteristic of fungal isolates obtained from home made cigarette
Fig: Fungal colonies isolated from home made cigarette Fig: Home made cigarette

10. SAMPLE LPCB OBSERVATION CHARACTERS INDENTIFIED
FUNGI
1 Septate, hyaline, acute angle
branching, tree or fern like branching,
conidial head biseriate, conidia was
in chain and detached
Aspergillus sp.
2 The budding stage of the isolates was
observed
Saccharomyces sp.
3 Bluish green with white border,
hyphae septate, hyaline. Conidio
phores,branched phialides group in
brush like clusters at the end of the
conidio phores, conidia unicellular,
round hyaline arranged in chain
Penicillium sp.
4
Non septate hyphae having branch
sporangio phores with sporangium at
terminal ends
Mucor sp.

11. SAMPLE LPCB OBSERVATION CHARACTERS INDENTIFIED
FUNGI
1 septate, hyaline, acute angle
branching, tree or fern like
branching, conidial head
biseriate, conidia was in chain
and detached.
Aspergillus sp.
2 Bluish green with white
border, hyphae septate,
hyaline.
Conidiophores,branched
phialides group in brush like
clusters at the end of the
conidio phores, conidia
unicellular, round hyaline
arranged in chain
Penicillium sp.
3
The budding stage of the
isolates was observed
Saccharomyces sp.

12. ISOLATE
NO
GRAM
STAINING
OBSERVATION
BIOCHEMICAL TEST IDENTIFIED
BACTERIA
Coag
ulase
test
Catala
se test
MR VP Indole Citrate
1.
+ve +ve +ve +ve -ve +ve Staphylococcus
aureus
2.
-ve -ve -ve +ve -ve +ve Streptococcus
pyogenes

13. MR
( - ve)
MR (+ ve) Citrate
(+ ve)
VP
(+ ve)
Coagulase test
Catalase test
Indole
(-ve)

14. Identification of Bacterial Isolates:
Isolates of bacteria obtained from bidi sample were
identified by observing the culture characteristics of
the colonies in media, gram staining and also by
performing biochemical test in accordance with
Bergey’s Manual of Determinative Bacteriology - 8th
ed. (Buchanan and Gibbons, 1974) and 9th ed. (Holt
et al., 1984).
 Identification of Fungal
Isolates:
Different fungal isolates were
obtained from the sample.
Identification was done by
observing the culture
characteristics of colonies in
media and also by staining
the isolates by LPCB method.
 Four isolates were obtained from bidi-
Aspergillus sp., Mucor sp., Saccharomyces
sp., Penicillium sp.
Three isolates obtained from home
made cigarette-
Aspergillus sp., Saccharomyces sp.,
Penicillium sp.
Two isolates were identified
from the sample-
Staphyloccocus aureus
and Streptococcus pyogenes.

15.  Components in fungal toxicants in
smoke block protein synthesis in
Macrophage
 Inhibits Tumor Necrosis Factor –α
signaling
 Activates Zipper Sterile Alpha
Motif kinase (ZAK), (Wong J.,
unpublished data, 2011)
Membrane Attached Phosphorylated
Kinases (MAPK) activate transcription
factors that lead the expression of
tumor genes. (Wong J et al., 2011)
Leads the Benign tumor in lungs and
Chronologically Pulmonary
Carcinoma.
(Proliferation of CD4 and Th 17 cell)
Storage in moist places
Air born microbes grew in crushed
Tobacco.
Collected toxicants consist of more number
of fungal colonies
Excreted toxin due to burn
Toxin and Nicotine inhaled by smoker
Pulmonary mycotoxicosis occur
Toxic mold exposed mycotoxicosis in
GIT and Sinuses.
Secondary bacterial infection occur
Chronic Obstructive Pulmonary Disease
(COPD) Occur

17.  According to my study different colonies of fungi and bacteria were
isolated and characterize from the sample. Bidi sample consist of more
fungal colonies then home made cigarette.
 Bacterial colonies were isolated from bidi. No bacteria was found in
home made cigarette.
 Fungal colonies isolated in this study were Aspergillus sp,
Saccharomyces sp, Mucor sp, Penicillium sp and bacterial colonies
isolated were Staphylococcus aureus and Streptococcus pyogens.
 Tobacco smoke is a bioaerosol that contain endotoxin, peptidoglycan
and various fungal constituents. This knowledge may help to explain
the high prevalence of respiratory disorders such as broncho alveolar
neutrophilia, air way obstruction and bronchial hypersensitivity among
smokers and individuals exposed to second hand smoke, since these
symptoms are also commonly associated with exposure to bio aerosols.
 Public awareness that tobacco smoke contains high concentration of
bacterial and fungal constituents may contribute to reduce smoking.

18.  B. Slutzker, G. Harmon, and P. Edmonds, “Microbiological content of
tobacco smoke,” The American Journal of the Medical Sciences, vol.
243, pp. 196–201, 1962.
 Larsson et al; license Bio Med Central ltd., Identification of bacterial
and fungal components in tobacco and tobacco smoke, 2008.
 Malson JL. et al; Pharmacol Biochem behave of Bidis –hand rolled,
Indian cigarette effects on physiological, biochemical and subjective
measure, 2002.
 Rahman M, et al; Public Health Bidi smoking and health, 2000.
 J. Forgacs and W. T. Carll; “Mycotoxicoses: toxic fungi in tobaccos,”
Science, vol. 152, no. 3729, pp. 1634–1635, 1966.

19. THANK YOU
Sincerest Gratitude to Supervisor :
Bhriganka Bharadwaj
Assistant Professor
Department of Microbiology
Assam down town University
THE END
Mail id. :
pdolakasaria@gmail.com
avranu132@gmail.com