Tuesday, November 10, 2015

Lab 5: Determination of Antimicrobial Effects of Microbial Extracts

Introduction


Bacteriocins are proteinaceous toxins produced by bacteria, it can inhibit the growth of closely or similar bacterial strains. It is also a naturally produced antimicrobial agent.  Bacteriocins can be found in numerous bacteria however, the ones that are produced by lactic acid bacteria (LAB) are significant to the food industry. This is because, they have huge potential application as one of the natural biopreservatives. As interest in natural and minimally processed food is in demand, more research is done to cater for consumer needs.

Lactic acid bacteria (LAB) are characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant, able to ferment carbohydrates for energy and lactic acid production. Thus, LAB bacteriocins can work via different mechanisms to exert an antimicrobial effect. 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 several microorganisms such as Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.


Objective



To determine the antimicrobial effects of extracellular extracts of selected LAB strains.

Material 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˚
CUV/V is spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract

Procedure 


(Refer to lab manual)


Results


Part I: Determination of Bacteriocin Activity Via Agar Diffusion Test

Absence of inhibition zone

Strains of LAB
Strains of spoilage/pathogenic bacteria
Inhibition zone(cm)
LAB 1
Staphylococcus aureus
No inhibition zone
LAB 2
Staphylococcus aureus
No inhibition zone

Part II: Determination of Bacteriocin Activity Via Optical Density
Reading of optical density from spectrophotometer

Dilution
OD600 of Spoilage/ Pathogenic Bacteria
Reading 1
Reading 2
Reading 3
Average
0x
0.421
0.408
0.396
0.408
2x
0.912
0.903
0.885
0.900
10x
1.037
1.005
1.024
1.022
50x
0.955
0.962
0.961
0.959
100x
0.908
0.946
0.824
0.893
Equation
y= - 0.0021x + 1.1
OD600 of control
0.233
0.231
0.224
0.229
50% of OD600
0.117
0.116
0.112
0.115
AU/ml
469.05

Discussion

Part I: Determination of Bacteriocin Activity Via Agar Diffusion Test


- Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains
- Bacteriocins are usually effective against gram-positive microorganisms.
-LAB bacteriocins may be inefficient to inhibit Gram-negative organisms because the outer membrane hinders the site for bacteriocin action, which is the cell membrane 
- Different mechanisms of action have been proposed for bacteriocins: alteration of enzymatic activity, inhibition of spore germination and inactivation of anionic carriers through the formation of selective and non-selective pores
- Staphylococcus aureus is a gram -positive coccal bacterium .Although it has thick cell wall made of polysaccharides and proteins but it is easily digested by acid particularly acetic acid produced by lactic acid bacteria (LAB).
-If there is a circle around the paper disk or colony of lactic acid bacteria (LAB), that means bacteriocin is able to inhibit the growth of Staphylococcus aureus . This is the zone of inhibition.
- The larger the inhibition zone means that the bacteriocins is effective on the pathogenic bacteria and vice versa.
-For our lab result, there is no inhibition zone. This is because not enough lactic acid bacteria (LAB) are being applied around the pathogenic bacteria. Without adequate numbers of lactic acid bacteria (LAB), the point of critical mass which is needed cannot occur and the bacteria will be unable to have the desired impact on the symptoms being treated.
-The bacteriocins produced by the lactic acid bacteria(LAB) is not strong as antibiotics.

Part II : Determination Of Bacteriocin Activity Via Optical Density

-The serial dilution of extracellular extract is done as shown in the table below.
- Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments resulting in concentration curves with a logarithmic scale.


0x (ml)
2x (ml)
10x (ml)
50x (ml)
100x (ml)
Control (ml)
EE
5
2.5
0.5
0.1
0.05
0
MRS
0
2.5
4.5
4.9
4.95
5
Total:
5
5
5
5
5
5

- Optical density, measured in a spectrophotometer, can be used as a measure of the concentration of bacteria in a 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 working with a particular type of cell, we would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will use cells that are in their mid-log phase of growth. Typically the OD600 is measured.
- One arbitrary (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 ; Thus x= (y-c)/m
When y= Z/2, thus x= (Z/2-c)/m
From the graph, m = - (1.1-0.893) ÷ 100 = -0.0021
From the graph, c = 1.1
Thus, y = -0.0021x + 1.1
When y = 0.115 , x = ( 0.115- 1.1 ) ÷ - 0.0021
                             x = 469.05
Thus, AU/ml = 469.05

-From the result, the graph shows that as the serial dilution increases, the optical density decreases. This shows that there is negative inhibition of the pathogenic bacteria. This might be caused by the human error. When we transfer the extracellular extracts into the mixture, we should take the supernatant instead of pellet, we should not shake the broth containing LAB cultures after centrifuge because this will mix the supernatant and pellet together.



-The control shows lower reading than all sample of dilution. This might be due to we didn’t shake the bottle of bacteria culture before pipetting especially for the control.
-The other reason is using distilled water during serial dilution. The distilled water is colourless. When the LAB is diluted with distilled water, the optical colour density will become very much lower compared to the normal colour density of  LAB. Thus, the results obtained is wrong. 
- Therefore, the peptone is suggested to replace with the distilled water in the serial dilution as the colour of peptone is quite similar with the culture. 
 -The actual graph should be a linear graph as shown in the diagram below.


-Actually there should be a  positive inhibition on the growth pattern of the bacteria. Generally, as the serial of dilution increases, the optical density will increase. It shows lactic acid bacteria (LAB) has strong microbial effect on the pathogenic bacteria which is Staphylococcus aureus.The high concentration of extracellular extract and bacteriocins result in low growth rate of bacteria.

Conclusion
The results shows the potential of Lactic Acid Bacteria (LAB) as biopreservatives against both Gram postive (Escherichia coli) and Gram negative (Staphylococcus aureus) bacteria. Bacteriocin was extracted by centrifuge method is to determine for its antagonistic activity. Lactic Acid Bacteria (lactobacilli) will produce various substances 
such as bacteriocin to inhibit the growth of pathogenic microorganisms. Lactic acid bacteria (LAB) also synthesize bacteriocins that have antimicrobial effects. 

References



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