Laboratory's
Information :
Laboratory's
Number - LAB A203
Laboratory
Title : DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS
Lecturer's
Name : Associate Professor Dr. Liong Min Tze
Laboratory
Assistant : Madam Najmah
Group
Members : Charles Ng Wai Chun, Siti Hawa Binti Ramli, Nuramirah Binti Ramlan
Date of
Laboratory : 27 October 2015
LAB 5 : DETERMINATION OF ANTIMICROBIAL EFFECTS
OF MICROBIAL EXTRACTS
Introduction
The use of bacterial interactions is a new way to limit the pathogenic
germs growth. Some groups of bacteria can produce antimicrobial substances with
the function in order to inhibit the growth of pathogenic and spoilage
microorganisms. Detection of antimicrobial substances produced by lactic acid
bacteria against the undesirable germs is the objective of this experiment.
Microbiological and biochemical methods were used to identify lactic acid
bacteria having an antimicrobial activity.
The production of antimicrobial agents such as organic acids,
hydrogen peroxide and bacteriocin or related substances from the extraction of
microbial bacteria plays a role in controlling both pathogenic and spoilage
microbe to grow. Since nowadays consumers demand “natural” and “minimally
processed” food, the interest in naturally produced antimicrobial agents such
as bacteriocins is on the rise of demand. The discovery of bacteriocins gave a
new way for food development in better hygienic quality as well as improve the
safety aspects for the consumption of the food. Bacteriocins are proteinaceous
compounds that mainly inhibit closely related species. Some bacteriocins have
been shown to possess the ability to inhibit the actions of unrelated genera
such as enteropathogenic bacteria and gram- negative bacteria. For these
reasons bacteriocins are promising candidates for natural bio-preservation
of food. Since nowadays consumers demand “natural” and “minimally
processed” food, the interest in naturally produced antimicrobial agents such
as bacteriocins is on the rise of demand. The discovery of bacteriocins gave a
new way for food development in better hygienic quality. Bacteriocins are
proteinaceous compounds that mainly inhibit closely related species. Some
bacteriocins have been shown to possess the ability to inhibit the actions of
unrelated genera such as enteropathogenic bacteria and gram- negative bacteria.
For these reasons bacteriocins are promising candidatesfor natural
biopreservation of food.
Lactobacillus bacteriocins are found within each
of the four major classes of antimicrobial proteins produced by lactic acid
bacteria. Lactobacilli produce many different bacteriocins of
similar activity. Different classes of lactic acid bacteria (LAB) bacteriocins
have been identified on the basis of biochemical and genetic characterization.
We sought to characterize bacteriocins for their structural properties and
determine their antimicrobial activities against some common human pathogens
including Gardnerella vaginalis, Pseudomonas aeroginosa, Proteus
vulgaris, Escherichia coli, Enterobacter cloacea, Streptococcus milleri,
Staphylococcus aureus and Candida albicans.
Objective
1) To determine the
antimicrobial effects of the extracelluar extracts of selected LAB strains.
2) To provide the
experience of using optical density spectrophotometer.
3) To provide experience
of preparing serial dilution.
Materials and Reagents
1) MRS
broth
2) Sterile filter paper disk
2) Sterile filter paper disk
3)
Sterile universal bottles
4) Cultures of LAB and spoilage/pathognic organisms
4) Cultures of LAB and spoilage/pathognic organisms
5) Bench-top
refrigerated centrifuge
6)
Incubator 37oC
7) UV/Vis spectrophotometer
8) Distilled deionized water
9) Trypticase soy agar
10) Brain heart infusion agar
11) Yeast extract
7) UV/Vis spectrophotometer
8) Distilled deionized water
9) Trypticase soy agar
10) Brain heart infusion agar
11) Yeast extract
Procedure
(refer to the laboratory
manual)
Results
Part 1 : Determination of bacterioacin activity via agar diffusion
test
 |
| Photo 1 : No inhibition of Staphylococcus aureus growth |
Part 2 : Determination of bacteriocin activity via optical density
 |
| Photo 2 : OD₆₀₀ of Staphylococcus aureus at different dilutions (3 readings of 0x, 2x, 10x, 50x, 100x and Control are from column 2 to 7 , row A, B and C) |
Table 1: Calculations of OD₆₀₀ based on
the result obtained
Dilutions
|
OD₆₀₀ of S. aureus
|
|||
Reading
1
|
Reading
2
|
Reading
3
|
Average
|
|
0x
|
0.663
|
0.533
|
0.624
|
0.607
|
2x
|
0.922
|
0.841
|
1.041
|
0.935
|
10x
|
0.788
|
0.811
|
0.833
|
0.811
|
50x
|
0.350
|
0.330
|
0.330
|
0.337
|
100x
|
0.349
|
0.268
|
0.281
|
0.299
|
OD₆₀₀ of control
|
0.217
|
0.211
|
0.219
|
0.216
|
50% of
OD₆₀₀
|
0.109
|
0.101
|
0.110
|
0.107
|
Graph 1: OD₆₀₀ of Staphylococcus aureus at
different dilutions
 |
Discussion
Part I: Determination of bacteriocin activity via agar diffusion
test
In this part, two strains of lactic acid bacteria (LAB) are used
which one of the strains labelled LAB 1 (8633) and the other strain
labelled LAB 2 (1515). The pathogenic organism used is Staphylococcus aureus.
From the result shown in the experiment that we conducted, there
is no inhibition zone seen around the paper disk dipped with both LAB 1 and LAB
2. This might be due to the inability of both LAB strains to inhibit the growth
of Staphylococcus aureus. Besides, it might be due to
the Staphylococcus aureus had
overcome the inhibition zone as it was still continuously growing on the petri
dish containing nutrient (BHI agar) when it was still in the incubator.
Part II: Determination of bacteriocin activity via optical density
In this part, LAB 2 (1515) and Staphylococcus aureus were
used in the experiment conducted.
Before conducting the experiment, LAB 2 was centrifuged and the it
was separated into two parts: the less dense part which floated at the upper
side of the liquid is the supernatant (containing extracellular extracts,
bacteriocin as a metabolic byproduct produced by LAB 2) and the denser pellet
which sedimented at the bottom of the liquid (LAB 2 cells). Heat will be
produced during the centrifugation process so the centrifugation was carried
out at a lower temperature as some of the cells are heat-sensitive.
 |
| Photo 3: Centrifugation of LAB. |
Serial dilution of the extracellular extracts of
LAB 2:
Dilutions
(ml) / Components
|
0x
|
2x
|
10x
|
50x
|
100x
|
Control
|
Extracellular
extract
|
5.00
|
2.50
|
0.50
|
0.10
|
0.05
|
0.00
|
MRS
|
0.00
|
2.50
|
4.50
|
4.90
|
4.95
|
5.00
|
MRS DS +Staphylococcus aureus
|
5.00
|
5.00
|
5.00
|
5.00
|
5.00
|
5.00
|
Total
|
10.00
|
10.00
|
10.00
|
10.00
|
10.00
|
10.00
|
 |
| Photo 4 : Universal bottles containing mixtures after serial dilutions. |
To
measure the optical dentistry of the pathogenic bacteria, a spectrophotometer
was used. The measurement is based on the absorbent of light with a specific
wavelength by the Staphylococcus aureus cells.
The wavelength of light used is 600 nm.
One arbitrary unit (AU) is
defined as the dilution factor of the extracellular extract that inhibited 50%
of the pathogenic bacteria growth and expressed as AU/ml. Based on Graph 1,
there is no AU/ml in this experiment because 50% OD₆₀₀ of
control = 0.107 does not intersect with the line of best fit of OD₆₀₀ at
different dilutions.
In actual case, the control of
the experiment supposes to show the highest OD₆₀₀ reading
among all the other dilutions as the growth of Staphylococcus
aureus is not inhibited by any bacteriocins produced by LAB and
so, it can grow when it is still in the incubator. Hence, the Staphylococcus aureus can absorb most of the light
which will produce the highest OD₆₀₀ reading). The
actual graph as in the normal case should be a linear graph with an increasing
of OD₆₀₀ value at higher dilution levels as the concentration of Staphylococcus aureus becomes
higher at higher extracellular extract dilution levels. However, the OD₆₀₀ readings
at different dilution levels in our experiment are also inconsistent, so we
could not get the result as in actual case.
This result could be caused by
the contamination of the extracellular extract (supernatant) with the LAB 2 (cell
pellets) due to the shaking of centrifugation tube when we handled it. So, the
control of the experiment actually consists of 5 ml of MRS and 5 ml of
extracellular extract that had been contaminated with LAB. Since the MRS is a
specific growth medium for LAB, LAB can grow well in the medium, which leaded
to the antimicrobial effects of Staphylococcus aureus by the LAB, resulting a poor absorbent of light by Staphylococcus aureus, and therefore, a lower
50% of OD₆₀₀ reading
than in the actual case.
Reference
1) Wikipedia, the
free encyclopedia (28 April 2015). Retrieved from
https://en.wikipedia.org/wiki/Antimicrobial
2) National Center for
Biotechnology Information, U.S. National Library of Medicine (2001),
Bacteriocins: safe, natural antimicrobials for food perservation. Retrieved
from http://www.ncbi.nlm.nih.gov/pubmed/11764886
3) Wikipedia, the
free encyclopedia (16 September 2015). Retrieved
from https://en.wikipedia.org/wiki/Bacteriocin
4) U.S. Department of
Health & Human Services (6 November 2015), Staphylococcus. Retrieved
from http://www.foodsafety.gov/poisoning/causes/bacteriaviruses/staphylococcus/
