INTRODUCTION
An antimicrobial is defined as any of a large variety of chemical compounds and physical agent that kills or destroys microorganisms or inhibits their growth by preventing their development. Antimicrobials can be classified according to their function. Agents that kill microbes are called microbicidal, while those that merely inhibit their growth are called biostatic. The use of antimicrobial medicines to treat infection is known as antimicrobial chemotherapy. The main classes of antimicrobial agents are disinfectants, which kill a wide range of microbes on non-living surfaces to prevent the spread of illness, antiseptics and antibiotics. The production and use of the antibiotic penicillin in the early 1940s became the basis for the era of modern antimicrobial therapy. Streptomycin was discovered in 1944, and since then many other antibiotics and other types of antimicrobials have been found and put into use. A major discovery following the introduction of these agents into medicine was the finding that their basic structure could be modified chemically to improve their characteristics.
Some members of LAB produce bacteriocins and bacteriocins-like substances which may inhibit growth of spoilage and pathogenic microorganisms. Bacteriocins from LAB are bioactive peptides or proteins with antimicrobial activity toward Gram positive bacteria, including closely related strains and/or spoilage and pathogenic bacteria. Bacteriocins are ribosomaly synthesized and extracellulary released bioactive peptides or peptide complexes which have bactericidal or bacteriostatic effect. Moreover, bacteriocins are innocuous due to proteolytic degradation in the gastrointestinal tract In view of its technological and biochemical properties the above bacteriocin can be considered as a potential biopreservative. Some of other LAB like Enterococcus, Lactococcus, and Pediococcus are also widely used as natural preservatives, due to the potential production of metabolites with antimicrobial activity such as organic acids, hydrogen peroxide, antimicrobial enzymes and bacteriocins.
OBJECTIVE
1. To determine the antimicrobial effects of extracellular extracts of selected LAB
strains.
2. To provide the experience of using optical density spectrophotometer.
MATERIALS AND REAGENTS
MRS broth
Sterile filter paper disk ( 50mm x 50mm )
Forceps
Sterile universal bottles
Cultures of LAB and spoilage / pathogenic organisms
Bench-top refrigerated centrifuge
Incubator 30°C and 37°C
UV / Vis spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract
PROCEDURE
PART 1 : Determination of bacteriocin activity via agar diffusion test
1. All petri dishes was labelled according to the spoilage organisms and strains of
LAB used.
2. Each plate was used for only one strain of spoilage organism and one strain of
LAB. The plate was divided into 4 parts, 2 parts for one member of the group and
each part for one replicate.
3. Each group had only one strain of LAB and one strain of spoilage/pathogenic
organism for this experiment.
4. 10ml of trypticase soy-yeast extract agar (TSAYE) was loaded into the labelled
petri dishes and the agar was made sure that it covered the entire surface of the
plate. The petri dishes were then left aside until the agar solidify.
5. 2 ml of the broth containing the spoilage organism, E. coli was inoculated into 10
ml of brain heart infusion agar (BHI) and vortexed.
6. The mixture was loaded on top of the layer of solidified TSAYE agar, making sure
that the mixture covered the entire surface and then left aside for solidification to
take place.
7. The broth containing LAB cultures was centrifuged. The supernatant was used as
the extracellular extracts.
8. A sterile filter paper disk was picked up aseptically with a pair of sterile forceps
and the disk of filter paper was dipped into the extracellular extract. The excess
extract has been drained off before proceeding.
9. The paper disk was placed on top of the solidified BHI agar which contained
spoilage organism.
10. All the plate prepared were incubated at 37 °C for 24 to 48 hours.
11. The inhibition zones were measured (in cm) and the result was recorded.
PART 2 : Determination of bacteria activity via optical density
1. The broth containing LAB culture (Lactobacillus plantarum) is centrifuged and the
supernatant is used as extracellular extract.
2. A serial dilution of extracellular extracts (0x, 2x, 10x, 50x and 100x) are prepared
with total of 5 ml for each serial dilution. A control medium is also prepared. The
extract are diluted with double-strength MRS broth with volume as shown,
Dilution Mixture
|
0x
|
2x
|
10x
|
50x
|
100x
|
Control
|
Extracellular Extract (ml)
|
5.0
|
2.5
|
0.5
|
0.1
|
0.05
|
0
|
MRS Broth (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
|
3. Each dilution is added with another 5 ml of double strength MRS broth and 1 ml of
culture containing spoilage pathogen (E. coli).
4. Each mixture are vortex to ensure uniform distribution of the mixture.
5. Each mixture are incubated at 37°C for 24 hours.
6. A negative-control for “auto-zero” are prepared via the spectrophotometer by
adding 200 μl of double strength MRS broth to the first chamber in the first column
of a 8 X 12 chamber well.
7. 200 μl of mixture are transferred to the first column of the well (control at the
second row, 0x at third row, 2x at fourth row, 10x at fifth row, 50x at sixth row and
100x at seventh row)
8. 100 μl of distilled water are placed in all remaining chamber.
9. The mixture are diluted by transferring 100 μl of the content in the first column to
the chamber at the second column of the same row. The content are mix
thoroughly before the process is repeated for other chamber. The remaining 100
μl at the last column are discarded.
10. The optical density of the E. coli in the mixture are measured with the
spectrophotometer.
11. The data are recorded and the column with reading between 0.1 and 0.4 are
selected.
12. The reading are subtracted with the reading of “auto-zero” before any
subsequent calculation.
13. A graph is plotted and the value of IC50 is determined from the graph.
RESULTS
PART 1 : Determination of bacteriocin activity via agar diffusion test
 |
| Figure 1 : Measuring the diameter of inhibition zone |
Strain of spoilage organism
|
Strain of LAB
|
Group member
|
Diameter of inhibition zone (cm)
| ||
Escherichia coli
|
Lactobacillus fermentum
|
LAB 1
|
LAB 2
|
Average
| |
Tay Yen Nee
|
0.78
|
0.84
|
0.81
| ||
Tang Hock Wei
|
0.84
|
0.88
|
0.86
| ||
Zahirah
|
0.80
|
0.86
|
0.83
| ||
Total average = 0.81 cm + 0.86 cm + 0.83 cm
= 2.5
Final average = 2.5 / 3
= 0.83
The conclusion here is that the inhibition zone is averagely 0.83 cm in diameter.
PART 2 : Determination of bacteria activity via optical density
 |
| Graph 1 : OD₆₀₀ of Staphylococcus aureus at different dilutions |
 |
| Figure 2 : OD₆₀₀ of Staphylococcus aureus at different dilutions |
Table 2: Calculations of OD₆₀₀ based on the result
Dilutions
|
OD₆₀₀ of S. aureus
| |||
Reading 1
|
Reading 2
|
Reading 3
|
Average
| |
0x
|
0.371
|
0.353
|
0.362
|
0.362
|
2x
|
0.551
|
0.569
|
0.608
|
0.576
|
10x
|
0.305
|
0.317
|
0.328
|
0.317
|
50x
|
0.880
|
0.858
|
0.871
|
0.870
|
100x
|
0.668
|
0.520
|
0.770
|
0.653
|
OD₆₀₀ of control
|
0.208
|
0.202
|
0.216
|
0.209
|
50% of OD₆₀₀
|
0.104
|
0.101
|
0.108
|
0.105
|
Discussion
PART 1 : Determination of bacteriocin activity via agar diffusion test
 |
| Figure 3 : 3 labelled petri dishes waiting to measure the inhibition zone |
For part 1, we ought to know that bacteriocin are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. Applications of bacteriocins are being tested to assess their application as narrow-spectrum antibiotics.
In this part of the experiment, two strains of lactic acid bacteria or also known as LAB throughout the whole report were used, which are LAB 1 ( 8633 ) and LAB 2 ( 1515 ) respectively. On the other hand, the patogenic organism used is Staphylococcus aureus. By following the procedures of the experiment, we get to observed only a little area of inhibition zone around the paper disk dipped with both LAB 1 and LAB 2. Theoratically, the larger the clear area around the filter disk, the more effective the bacteriocin. But the throughout the experiment, only one out of three culture plates that shows a significant inhibition zone. The other two which didn’t shows a significant zone might be due to the inability of both LAB 1 & 2 strains to inhibit the growth of Staphylococcus aureus.
PART 2 : Determination of bacteria activity via optical density
 |
| Figure 4 : Universal bottles containing mixtures after serial dilutions. |
 |
| Figure 5 : Transferring using a pipette |
For part 2 of this experiment, we have to determine the bacteria activity via optical density by using the help of a laboratory instrument called a spectrophotometer. But first, we have to understand optical density. Optical density is the degree to which a refractive medium retards transmitted rays of light and is measured using a spectrophotometer. A spectrophotometer is included in the field of Spectrophotometry. Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance. Therefore, a spectrometer can be defined as an apparatus for measuring the intensity of light in a part of the spectrum, especially as transmitted or emitted by particular substances. 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 working with a particular type of cell, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will want to use cells that are in their mid-log phase of growth. Typically the OD600 is measured. This means that the wavelength of light used is 600 nm. Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications.
An arbitrary unit is a relative unit of measurement to show the ratio of amount of substance, intensity, or other quantities, to a predetermined reference measurement. From the experiment, an arbitrary unit ( AU ) is defined as the dilution factor of the extracellular extract that inhibited 50 % of the spoilage / pathogenic bacteria growth and expressed in AU/ml. But unfortunately based on graph 1 that we plotted, there is no AU/ml that can be obtained 50% OD₆₀₀ of control 0.105 does not intersect with the line of best fit of OD₆₀₀ at different dilutions.
For this part of the experiment, the actual graph 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. But unfortunately it didn’t appear like the result we wanted which is a linear graph . This may be caused by contamination of the extracellular extract with the LAB 2 because we fail to handle it while shaking the centrifugation tube.
CONCLUSION
Some of the microorganisms can synthesis certain substances that have antimicrobial effects.
REFERENCE
5 . http://people.hofstra.edu/beverly_clendening/adv_molecular_biology/Protocols/Measuring_Optical_Dens.html
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