Lab
5: Determination of Antimicrobial Effects of Microbial Extracts
Introduction
Antimicrobial is an agent which kills
microorganisms and inhibit their growth. The production of antimicrobial agents
such as organic acids, hydrogen peroxide and bacteriocins from the extraction
of microbial bacteria plays an important role in controlling both pathogenic
and spoilage microorganisms. Bacteriocins have a large and diverse group of
ribosomally-synthesized antimicrobial proteins with killing mechanism. Bacteriocins
are produced by lactic acid bacteria (LAB). Bacteriocins also inhibit the
growth of Staphylococcus aureus and Escherichia coli (E.coli).
Lactic acid bacteria (LAB) are numerous in
number and play a vital role in conversion of raw materials into lacto-fermented
food. The shape of LAB is rod-shaped or spherical. It is non-sporulating,
non-respiring and lack of catalase Gram-positive bacterium. It is also acid-tolerant
which has low pH range. LAB commonly produce antimicrobial substances with
activity against the homologous strain. LAB often produce microbicidal
substances with effect against gastric and intestinal pathogens.
Staphylococcus aureus is
a non-motile and non-spore forming facultative anaerobic Gram-positive coccal
bacterium. It is a non-pathogenic bacterium but it causes skin infections,
respiratory disease and food poisoning. It looks like grape-like clusters and
has large, round, golden-yellow colonies. It reproduces asexually by binary
fission. It is a catalase-positive which mean that it can produce enzyme
catalase. It undergoes fermentation of glucose and produces lactic acid.
Escherichia coli (E.coli) is
a non-sporulating, Gram-negative and rod-shaped bacterium. E. coli are a
large and diverse group of bacteria. It
is a facultative anaerobic bacterium. Most E. coli strains are harmless, but
some serotypes can cause serious food poisoning or relatively brief diarrhea in
hosts and are occasionally responsible for product recalls due to food
contamination. Under favourable conditions, it takes only 20 minutes to
reproduce. It is able to survive outside the body of a host for a limited
amount of time.
Objective
·
To learn the way of
measuring optical density by using spectrophotometer.
·
To determine
antimicrobial effects of extracellular extracts of selected LAB strains.
·
To study the method of
preparing serial dilution.
Materials and reagents
·
Trypticase soy agar
(TSAYE)
·
Bacteria strains
·
Brain heart infusion agar
(BHI)
·
Culture of lactic acid
bacteria (LAB)
·
Pathogenic organisms
·
MRS broth
·
Distilled deionized water
·
Petri dish
·
Incubator
·
Pipette
·
Forceps
·
Sterile filter paper disk
(50mm x 50mm)
·
Sterile universal bottles
·
Bench-top refrigerated
centrifuge
·
UV/Vis spectrophotometer
Procedure
Part I: Determination of bacteriocin
activity via agar diffusion test
1. Three
petri dishes were labelled for each group.
2. A
petri dish was shared between two person.
3. Each
petri dish was divided into four parts and each side for one replicate.
4. A
strain of pathogen (Staphylococcus aureus)
and a strain of lactic acid bacteria (LAB) were used in this experiment.
5. 10
ml of trypticase soy-yeast extract agar (TSAYE) was added into each labelled
petri dish and the petri dish was moved in “8 shape” on the work surface to
ensure the agar was distributed evenly.
6. The
petri dish was left aside for solidification.
7. 2
ml of pathogen (Staphylococcus aureus)
was innoculated into 10 ml of brain heart infusion (BHI) agar.
8. The
innoculation process must be carried out quickly to avoid solidification of BHI
agar.
9. The
mixture is then loaded on top of the TSAYE agar layer and the petri dish was
moved in “8 shape” on the work surface to ensure the agar was distributed
evenly.
10. The
petri dish was left aside for solidification.
11. 50
ml of broth containing LAB cultures was centrifuged at 3500rpm for 10 minutes.
The supernatants will be used as extracellular extracts.
12. The
forceps were held by its handle and the tip of the forceps were heated with the
flame of a Bunsen burner.
13. A
sterile filter paper disk was aseptically picked up using sterile forceps.
14. The
paper disk was then dipped into Lactobacillus Fermentum containing
extracellular extracts. The excess extract was drained off completely.
15. The
paper disk was placed on top of the solidified BHI agar.
16. The
petri dishes were incubated for one day at 37°C.
17. Upon
incubation, the inhibition zones were measured (in cm) and the readings were
recorded.
Part
II: Determination of bacteriocin activity via optical density
1. Universal
bottles were prepared.
2. Each
of the universal bottle was labelled with 0x, 2x, 10x, 50x ,100x and control.
3. Lactobacillus Plantarum
was mixed with MRS to be diluted to final volume of 5 ml in each universal
bottle labelled. The volume of the Lactobacillus Plantarum and MRS needed for
each universal bottle is shown as below:
0x
|
2x
|
10x
|
50x
|
100x
|
control
|
|
Volume
of Lactobacillus
Plantarum (ml)
|
5.0
|
2.5
|
0.5
|
0.1
|
0.05
|
0
|
Volume
of MRS (ml)
|
0
|
2.5
|
4.5
|
4.9
|
4.95
|
5.0
|
Total
(ml)
|
5.0
|
5.0
|
5.0
|
5.0
|
5.0
|
5.0
|
4. Each
of the mixture was then added with 5 ml of MRS and 1 ml of pathogen Escherichia coli (E.coli).
5. The
universal bottles were then incubated for 12-15 hours at 37°C.
6. Upon
incubation, the optical density of the pathogenic bacteria in each universal bottle
was measured at 600nm using spectrophotometer.
Results
Part I: Determination of bacteriocin
activity via agar diffusion test
Strain of LAB
|
Strain of pathogenic bacteria
|
Inhibition
zone (cm)
|
||
Lactobacillus Fermentum
|
Staphylococcus aureus
|
Diamater1 (cm)
|
Diameter 2 (cm)
|
Average diameter (cm)
|
0.75
|
0.80
|
0.78
|
||
0.67
|
0.70
|
0.69
|
||
0.73
|
0.76
|
0.75
|
||
0.60
|
0.64
|
0.62
|
||
0.72
|
0.75
|
0.74
|
||
0.76
|
0.70
|
0.73
|
||
Part II: Determination of bacteria
activity via optical density
Dilutions
|
OD600 of spoilage/ pathogenic bacteria
|
Strain 1 : E.coli
|
|
0x
|
0.254
|
2x
|
0.384
|
10x
|
0.351
|
50x
|
0.349
|
100x
|
0.428
|
Equation
|
y=0.00098x+0.33
|
OD600 of control
|
0.1370
|
50% of OD600
|
0.0685
|
AU/ ml
|
-266.84
|
Discussion
Part I: Determination of bacteriocin
activity via agar diffusion test
Bacteriocins are ribosomally-synthesized
peptides or protein toxins produced and released by
lactic acid bacteria (LAB) to inhibit the
growth of similar bacteria. The
antagonistic potential of LAB may also involve the production of bacteriocins
which are active against Staphylococcus
aureus. They generally have an inhibitory or even a bacteriolytic effect on
target species.
Brain-heart infusion agar (BHI) is a highly nutritious general-purpose growth medium for
culturing fastidious and non-fastidious microorganisms. It is made by boiling
cow or porcine hearts and brains. Antibiotic
sensitivity is the susceptibility of bacteria to antibiotics. BHI agar
is often used in antibiotic sensitivity tests. Antibiotic susceptibility
testing (AST) is usually carried out to determine which antibiotic will be most
successful in treating a bacterial infection.
Trypticase
soy-yeast extract agar (TSAYE) are growth media for the culturing of bacteria.
They are general-purpose media, nourishment providing enough nutrients to allow
for a wide variety of microorganisms to grow. TSAYE contains enzymatic digests
of casein and soybean meal, which provides amino acids and other nitrogenous
substances.
Agar diffusion test is a test to determine
whether bacteria are affected by antibiotics. In this test, paper disk with LAB
are placed on an agar plate where bacteria have been placed, and the plate is
left to incubate. If LAB stops the bacteria from growing or kills the bacteria,
there will be an area where the bacteria have not grown enough to be visible.
This is called a zone of inhibition. The size of this zone depends on how
effective LAB is at stopping the growth of bacterium.
Part
II: Determination of bacteria activity via optical density
The
optical density (the degree to a refractive medium retards transmitted rays of
light) can be measured using a spectrophotometer. Optical density (OD) can be
used as a measurement of concentration of bacteria in a suspension. The light
is scattered when a visible light passes through a cell suspension. The greater
the light scatter, the higher the amount of bacteria and other material
present.
In this experiment, the cells at the
log phase of growth are used. This is because this phase of population growth
indicates that the cells are dividing at the most rapid rate, not overcrowded
and generally well nourished. Furthermore, OD600 is measured with a
spectrophotometer set at a wavelength of 420-660nm during the experiment. High
optical density and low transmittance leads to the high protection factor by a
filter.
Arbitrary unit (AU) or procedure defined unit is
a relative unit of
measurement to show the ratio of amount of substance, intensity, or other
quantities, to a predetermined reference measurement. In this experiment, arbitrary
unit is the dilution factor of the extracellular extract in which the 50% of
pathogenic bacteria growth is inhibited and is expressed as AU/ml. Thus, the
equation
is used to plot the graph.
The OD 600 of the positive control was then measured in
order to investigate the present of inhibition of pathogenic bacteria. From the
result above, the graph of the E. Coli shows positive gradient. This indicates
the positive inhibition on the growth pattern of the bacteria. Generally, the
OD value is higher for highly diluted solutions. It shows lactic acid bacteria
(LAB) have the stronger antimicrobial effect on the pathogenic bacteria from
highly diluted solution. The high concentration of extracellular extract and
bacteriocin result in low growth rate of bacteria.
Conclusion
Bacteriocins produced by lactic acid bacteria (LAB) as bio
preservatives against both Escherichia coli (E.coli) and Staphylococcus aureus. Lactic acid bacteria (LAB)
also synthesize bacteriocins that have antimicrobial effects. The
graph showing a positive gradient indicates positive inhibition of E.coli
result in high optical density.
Reference
