LAB5: Determination of Antimicrobial Effects of Microbial Extracts

Introduction

Pathogenic and spoilage microorganisms had created many undesired effects to human race. Antimicrobials are used to overcome these problems. Antimicrobial is a group of drugs includes antibiotics, antifungals, antiprotozoals, and antivirals that are used to destroy microorganisms or to prevent their development. They are used in various aspects such as in agriculture, surgery, treatment and food production. Some examples of antimicrobials which is critical important to human are Penicillins, Aminoclycosides and Streptogramins.

Lactobacillales or lactic acid bacteria (LAB) are microorganisms that produce lactic acid and proteinaceous bacteriocins which inhibit other microorganisms to survive. They are gram-positive, acid-tolerant, non-sporeforming cocci, coccobacilli or rods and lack catalase. All LAB grow anaerobically, but unlike most anaerobes, they can grow in the presence of O₂ as "aerotolerant anaerobes". LAB bacteriocins contribute to the taste and texture of fermented products and inhibit food spoilage bacteria by producing growth-inhibiting substances and large amounts of lactic acid as acidification inhibits the growth of spoilage agents. They are mainly used in food processing such as making yogurt, cheese, cultured butter, sour cream and sausage. Some examples of Lactobacillales are Lactobacillus, Lactococcus and Streptococcus.

Objective

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

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

UV/Vis spectrophotometer

Distilled deionized water

Trypticase soy agar (TSAYE)

Brain heart infusion agar (BHI)

Yeast extract

Procedure

(Refer to lab manual)

Results

Part 1: Determination of bacteriocin activity via agar diffusion test

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

Part 2: Determination of bacteriocin activity via optical density

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain : Staphylococcus aureus
	Reading 1	Reading 2	Reading 3	Average
0x	0.453	0.445	0.476	0.458
2x	0.671	0.665	0.673	0.670
10x	0.970	1.014	1.004	0.996
50x	1.090	0.995	0.950	1.012
100x	0.887	0.948	0.945	0.927
Equation				y = 0.0031x + 0.7115
OD600 of control	0.247	0.225	0.229	0.234
50% of OD600	0.124	0.113	0.115	0.117
AU/ml				-191.774

Discussion

Part 1: Determination of bacteriocin activity via agar diffusion test

From this experiment, the volume of the LAB extracellular extract and MRS needed for each universal bottle is shown as below:

Dilution Mixture	0x	2x	10x	50x	100x	Control
LAB 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

Antimirobials are the substance that produced by a certain microorganism to prevent other microorganisms to compete resources with them. In this experiment, bacteriocin, lacic acid, diacetyl and hydrogen peroxide act as the antimicrobials which is produced by Lactic Acid Bacteria (LAB) are tested by antimicrobial susceptibility testing to test their ability to inhibit the growth of SA (Staphylococcus aureus) culture. The goals of antimicrobial susceptibility testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. The effectiveness of LAB antimicrobials towards the growth of SA can be determined by measuring the inhibition zones around the LAB-staining paper disks. Inhibition zones are the places where the growth of a certain microorganism is inhibited by antimicrobial. The larger the inhibition zones, the higher the degree of sensitivity of SA to LAB antimicrobial.

However, in our experiment, the inhibition zones around the LAB paper disks cannot be determined as there is no growing of SA culture. This is probably caused by the concentration of SA spoilage culture is too dilute. In our experiment, we mixed 2 ml of SA culture into 100 ml of BHI agar but refer to our lab manual, we should mix 20 ml of SA culture into 100 ml of BHI agar. The low concentration of SA culture causes their growth in agar is totally suppressed by LAB antimicrobials. This results we cannot measure the degree of sensitivity of SA to LAB antimicrobial. Thus, we can at least speculate there is a suppression of LAB to SA culture as SA cannot grow in the agar with LAB. Besides, the bacteriocins produced by the lactic acid bacteria(LAB) may not strong enough.

Part 2: Determination of bacteriocin activity via optical density

Optical density can be measured by using the spectrophotometer. Optical density is used as a measure of the concentration of bacteria in a suspension. The spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium; it is used to determine the amount of light scatter. When visible light passes through a cell suspension, the light is scattered. Greater degree of scatter proves that more bacteria are present.

A spectrophotometer can be set at a wavelength of 420-660nm. Normally the OD600 is measured. OD600 is an abbreviation indicating the optical density of a sample measured at a wavelength of 600 nm. OD600 is also preferable to UV spectroscopy when measuring the growth over time of a cell population. It is because at this wavelength, the cells will not be killed as they would under too much UV light.

spectrophotometer

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.

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 experiment, the strain we used is Staphylococcus aureus. It is a gram-positive coccal bacterium that is frequently found in the respiratory tract and on the skin.

The result we obtained shows that the optical density decreases slightly when the dilution is 100 x. However, the graph still shows the positive gradient. This indicates the positive inhibition on the growth pattern of the bacteria. It means the higher the concentration of extracellular extract, the higher concentration of bacteriocin, the lower the growth rate of bacteria. The inaccurate result might due to human error, we did not use the pipette correctly that causes the volume of MRS and Staphylococcus aureus taken incorrect. Besides, when we transfer the extracellular extracts into the mixture, we should take the supernatant instead of pellet, if not it might affect the accuracy of result. The expectation of the result is when the serial dilution increasing, the optical density increasing too. The graph plotted should be in linear positive gradient.

Conclusion

LAB is used to produce bacteriocin which can inhibit the growth of bacteria (antimicrobial effect). Nowadays, the usage of LAB is getting attention as it exerts a strong antagonistic activity against many microorganisms, including food spoilage organisms and pathogens. The graph of determination of bacteria activity via optical density showing a positive gradient indicates positive inhibition of Staphylococcus aureus results in high optical density.

References

https://en.wikipedia.org/wiki/OD600

http://www.ifrj.upm.edu.my/20%20(01)%202013/17%20IFRJ%2020%20(01)%202013%20Noordiana%20(017).pdf

http://cid.oxfordjournals.org/content/49/11/1749.full