This document summarizes a student's honors thesis presentation on isolating and characterizing lactic acid bacteria from the gastrointestinal tracts of local pigeons in Bangladesh. The student collected samples from pigeon crops, gizzards, and intestines. Five samples were selected and cultured on MRS agar to isolate lactic acid bacteria colonies. The isolated bacteria were subjected to morphological, biochemical, and antibiotic resistance testing to characterize them. Based on microscopy and test results, it is assumed that two isolates are Lactobacillus species and three are Lactococcus species. The student concluded the study provides initial steps for potential use of the isolates as probiotic cultures.

1. Probiotic Profiling of Lactic Acid Bacteria from Local
(Bangladeshi) Pegions’ GIT
B. Sc. Honours Thesis Presentation
11 December, 2018
Lutfee Alom Brishti
Examination Roll No: 140636
Registration No: 1406225
Supervisor
S. M. Khaledur Rahman
Assistant Professor

2. CONTENT
Introduction
Probiotics
Aim and objectives
Health benefits of probiotics
Methods and procedures
Results
Discussion
Conclusion

3. INTRODUCTION
This study is about to isolate Lactic Acid Bacteria from
local pegion’s GIT
A microorganism which, when administered in adequate
amounts, confers a health benefit on the host
Gastrointestinal tract or GI tract begins at the mouth,
includes several important organs, and ends at the cloaca

4. PROBIOTICS
Derived from the Greek language meaning "for life"
Fuller in 1989 redefined probiotics as a live microbial
feed supplement which beneficially affects the host
Most common microorganisms found in probiotics
currently available are lactic acid bacteria especially
Lactobacillus and Bifidobacterium species
These resident microflora in the GIT improving its
intestinal microbial balance

5. AIM AND OBJECTIVES
Isolation and charectarization of Lactic Acid Bacteria from
local pegion’s GIT
Goal of this reserch is to gain scientific and technological
knowledge about Lactic Acid Bacteria from local pegion’s
GIT

6. HEALTH BENEFITS OF PROBIOTICS
Probiotics help balance in digestive system
Probiotics can prevent colorectal cancer
Probiotic supplements improve mental health conditions
Probiotics can help reduce symptoms of certain
digestive disorders
Probiotics may help one’s lose weight and belly fat

7. METHODS & PROCEDURES
Sample collection and sampling
Two local pegions with different age and color collected
from Borobazar,Palbari,Jessore
From two pegions’ GIT six samples (crop, gizzard,
intestine) were collected
Then stored in autoclaved plastic vials at -20⁰C in a
refrigerator
From those 5 samples were selected for further study

8. Media preparation
According to De Man et al, 1960; MRS Agar and Broth
designed to encourage the growth of lactic acid bacteria
MRS formulation was developed by de Man,Rogosa and
Sharpe
Media is selective for lactobacilli
Selectivity can be altered by pH adjustment

9. Distill water
MRS broth powder
Heat and agitate
Sterilize in autoclave
at 1210C at pressure
15 psi for 45 minutes
MRS agar powder
Media
preparation
Liquid
Solid
Cool at room
temperature
Poured into test
tubes/petridish
es

10. Protocol for isolation of lactic acid bacteria
One gram of sample dissolved in 9ml of 0.15% peptone
water solution
Dilluted up to ten logarithmic (10^-10) fold, then
incubated for 48 hours at 37oC into MRS agar plate
Typical LAB characteristics colonies were randomly picked
up and purified by streaking on fresh MRS agar plates
 During the tests, culture was kept in MRS agar stabs at
4⁰C temperatures

11. BACTERIAL CHARACTERIZATION (MORPHOLOGICAL TEST)
Gram staining Protocol
According to the protocol of Erkus (2007)
Single
colony
taken onto a
glass slide
Mixed with
distilled
water
Application
of cristal
violet
Application
of iodine
Alcohol
wash
Application of
safranin
Identifying gram
positive/negative

12. Catalase test
Conducted to determine the presence of the enzyme catalase
Catalase enzyme was found in most bacteria
If enzyme present, it would break hydrogen peroxide (H2O2)
and release of free oxygen

13. Protocol
According to ‘Amrita Virtual Lab Collaborative Platform’
A clean glass slide was divided into two sections with lubricant
pencil
One should be labeled as test and the other as control
Colony from
subculture smeared
onto glass slide
Hydrogen
peroxide
Oxygen bubble
produce/catalase
positive
Oxygen bubble
omit/catalase
negative

14. BIOCHEMICAL TEST
NaCl tolerance test protocol
According to Hoque et al., 2010 determination of NaCl tolerance of
isolated LAB
7 test tubes containing MRS broth adjusted with different concentrations
(5-10%) of NaCl
After sterilization, each test tube inoculated with 1% fresh overnight
culture of LABs and
Incubated at 37°C for 24 hours
After incubation, growth were determined by observing turbidity

15. pH tolerance test Protocol
Following Bates, Roger G. Determination of pH: theory and
practice. Wiley (1973) to test pH tolerance
Broth having pH 2.2 and pH 6.6 inoculated with 1% bacterial
suspension at 370C for 24 hours
Then samples were taken at 0, 10, 12, 14,16, 18 and 20 hours and
after 36 hours for cell resistance detection
Non-inoculated broth medium were served as control at pH 2.2
and pH 6.6

16. Bile salt tolerance test protocol
This test using protocol by Graciela and Maria (2001) with some
modifications, Zinedine and Faid (2007)
MRS broths with different concentration (0.15% and 0.3%) of bile Oxgall
used to determine the tolerance and growth rate of isolated LABs
Final pH of the medium adjusted to 6.5 and autoclaved at 1210C
Then 1% overnight cultured isolates of LABs were inoculated into the
MRSO (MRS-Oxgall) broth medium and Incubated at 370C for 24 hours
Survival rates of the isolates measured by Spectrophotometer
Non-inoculated MRS broth served as control

17. Antimicrobial activity
Antagonistic test are formed by well diffusion methods (Balcazar et al.,
2008; Cappuccino and Sherman, 2002)
Vibrio cholera, Escherichia coli, Klebsiella Pneumonia pathogens used
Pathogenic bacteria and isolates culture individually in nutrient broth
and incubated at 370C for 24 hour.
Nutrient
agar
Distilled
water
Media
preparation
Dissolved and
pourd into
petridishes
Pathogens spread
onto petridishes
Gell cutted by
gellcutter and
sample placed

18. Antibiotic resistance test
According to the standard agar diffusion method known as Kirby Bauer (Barry
and Thornsberry, 1985)
Nutrient agar Distilled water
Dissolved then
poured into
petridishes
Media cutted by
gellcutter and
antibiotic disk placed
Isolates spreaded
Media
prepared by

19. RESULTS
Isolation of LAB
S1D2
Figure: Lactic Acid Bacteria colonies on MRS agar plate (mother culture)
S2D3

20. Figure: Lactic Acid Bacteria colonies on MRS agar plate (mother culture)
S4D2
S5D3
S3D2

21. Figure : Lactic Acid Bacteria colonies on MRS agar plate (subculture)

22. Figure : Lactic Acid Bacteria colonies on MRS agar plate (subculture)

23. Gram staining

24. Figure : Gram stained smear observed under light microscope

25. Catalase test

26. Figure : Catalase test (positive/negative)

27. NaCl tolerance test

28. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 10 20 30 40 50 60
OD6O0nm
Time (hr)
PH 6.6
S1 S2 S3 S4 S5
pH tolerance test

29. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 10 20 30 40 50 60
OD6O0nm
Time(hr)
pH-2.2
S1
S2
S3
S4
S5
Figure : Growth curve of pH tolerance test of five isolates

30. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 10 20 30 40 50 60
OD-6O0nm
Time(hr)
0.15% Bile salt
S1
S2
S3
S4
S5
Bile salt tolerance test

31. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 10 20 30 40 50 60
0.3% Bile salt
Series1 Series2 Series3 Series4 Series5hour
Figure : Growth curve of bile salt tolerance for five isolates

32. Figure : Antimicrobial activity of five isolates
Antagonistic test

33. Antibiotic resistance test

34. Figure : Antibiotic resistance of isolates in nutrient agar

35. DISCUSSION
5 isolates from 6 samples remain at the end of the isolation
Among the 5 strains three are appeared as gram positive, catalase
positive , white;
Two were gram negative, catalase negative, white; Two were rod
and Three were cocci shaped
In this study assume that two rod shaped belongs to Lactobacillus
By analysis of microscopy and biochemical characterization,
it is assumed that another three isolates may be Lactococcus spp

36. CONCLUSION
I completed my study successfully and found a hopeful result
In this study the first step was taken to use the isolates as
cultures
Then I performed some test to determine probiotics
In future, this study can be broaden for probiotic products
production for higher range
This study can be continued to making probiotics more available

37. Thank you