LAB 5 : DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS

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

3) Sterile universal bottles
4) Cultures of LAB and spoilage/pathognic organisms

5) Bench-top refrigerated centrifuge

6) Incubator 37^oC
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/