Lab 5:
Determination of Antimicrobial Effects of Microbial Extracts
Introduction
Certain group of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage micro-organisms. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Interest in naturally produced antimicrobial agents, such as bacteriocins, is on the rise, since nowadays consumers demand “natural” and “minimally processed” food.
Bacteriocins comprise a large and diverse group of ribosomally synthesized antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those produced by lactid acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry biopreservatives. Different classes of LAB bacteriocins have been identified on the basis of biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Listeria monocytogenes, Staaphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
Part 1: Determination of bacteriocin activity via agar diffusion test
The agar diffusion test, or the Kirby-Bauer disk-diffusion method, is a means of measuring the effect of an antimicrobial agent against bacteria grown in culture.
The bacteria in question are swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.
Part 2: Determination of bacteriocin activity via optical density
Optical density, measured in a spectrophotometer can be used as a measured of the concentration of bacteria in suspension. As visible light passes through a cell suspension the light is scattered. Greater scatter indicates that more bacteria or other material is present. The amount of light scatter can be measured in a spectrophotometer. Typically, when we are working with a particular type of cell, we would determine the optical density at a particular wavelength that correlates with the phase of growth. Typically the OD600 is measured.
Objective
To determine the antimicrobial effects of extracellular extracts of selected LAB strains.
Result
Part 1: Determination of bacteriocin activity via agar diffusion test
Calculations
Inhibition zone:
Serial dilutions of extracellular extract
Y axis: Abs600 or OD600 X axis: Serial dilutions of extracellular extract
m and c: Constants
One arbitrary unit (AU) is defined as the dilution factor of the extracellular extract that inhibited 50% of the spoilage / pathogenic bacteria growth and expressed as AU/ml
Control: Abs600 = Z. Thus, 50% of Z = Z/2
Y = mx + c. Therefore, x = (Y – c)/m
When Y = Z/2, thus, x= (Z /2-c)
Strains of LAB
|
Strains of spoilage/pathogenic bacteria
|
Inhibition zone (cm)
|
Lp
|
Kp
|
0.7
|
Sa
|
-
| |
Pa
|
0.6
| |
Lb
|
Kp
|
0.7
|
Sa
|
-
| |
Pa
|
0.8
| |
Lc
|
Kp
|
0.6
|
Sa
|
-
| |
Pa
|
0.7
|
Strain of LAB
|
Strains of spoilage/pathogenic bacteria
|
Inhibition zone (cm)
|
LAB species
|
Sa
|
-
|
Kp
|
1.0
| |
Pa
|
1.05
|
Part 2: Determination of bacteriocin activity via optical density
Serial dilution of extracellular extract
Dilutions
|
OD600 of spoilage/pathogenic bacteria
| ||
Strain 1:Sa
|
Strain 2:Kp
|
Strain 3:Pa
| |
0x
|
-
|
-
|
-
|
2x
|
0.735
|
0.637
|
0.710
|
10x
|
1.032
|
0.691
|
0.076
|
50x
|
0.621
|
0.641
|
0.804
|
100x
|
0.502
|
0.765
|
0.528
|
Equation
|
y=-0.111x + 1.0
|
y=0.033x + 0.6
|
y=0.018x + 0.484
|
OD600 of control
|
0.270
|
0.829
|
0.432
|
50% of OD600
|
0.135
|
0.415
|
0.216
|
AU/mL
|
7.793
|
5.606
|
14.889
|
~Sa~
Dilutions
|
Abstract
|
0x
|
0.000
|
2
|
0.735
|
10x
|
1.032
|
50x
|
0.621
|
100x
|
0.502
|
~Kp~
Dilution
|
Abstract
|
0x
|
0
|
10x
|
0.637
|
20x
|
0.691
|
50x
|
0.641
|
100x
|
0.765
|
~Pa~
Dilutions
|
Abstract
|
0x
|
0
|
10x
|
0.710
|
20x
|
0.076
|
50x
|
0.804
|
100x
|
0.528
|
Discussions
Part 1: Determination of bacteriocin activity via agar diffusion test
Lactic Acid Bacteria (LAB) is Gram-positive, non-cocci, coccobacilli or rods with a DNA base composition of less than 53mol% G+C. They generally are non respiratory and lack catalase. This trait has follow throughtout the history, linked LAB with food fermentations, as acidification inhibits the growth of the spoilage agents.
Although they lack catalase, they possess superoxide dismutase and have alternative means to detoxify peroxide radicals, generally through peroxidase enzymes. The extract of lactid acid bacteria are gave inhibitions zone onto the indicators pathogen strains tested. This is an area around a paper disk or colony of bacteria or mold where no other organisms are growing.
If you are testing antibiotic sensitivity for example, you can impregnate paper disks with antibiotic and then put them on an agar plate of growing bacteria. The antibiotic then diffuses into the agar away from the disk. If the bacteria are sensitive to the antibiotic, they will not grow near the disk. The size of the zone is proportional to how sensitive the organism is. If the organism is resistant to the antibiotic, it will grow right up to the disk.
Part 2: Determination of bacteriocin activity via optical density
Optical density that is measured in the spectrophotometer can be used as the measurement of the concentration of the bacteria suspension. It is basically used for the measurement of transmittance or reflectance of solution that are depending on their wavelength.
Optical density of this experiment contain MRS medium and pathogenic bacteria was determined by using the spectrophotometer of OD600. One from the others type of LAB used is Lactobacillus plantirum. While the tree type of pathogenic microorganism that had been used were S.Aureus, P.Aeruginosa and K.Pneumonia.
The positive-controls that show higher optical density than the other sample show antimicrobial effect. While the negative-control was prepared in MRS with 2mL distilled water is for auto-zero via spectrophotometer.
Conclusions
Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. They are typically considered to be narrow spectrum antibiotics, though this has been debated. They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse.
References
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