Lab Report 5 by Chai Soo Teng

Name        :  Chai Soo Teng
Matric No :  111356

Lab 5:Determination of Antimicrobial Effect of Microbial Extracts

Introduction
          Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage micro-organisms.Organic acids,hydrigen peroxide,diacetly 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 synthesised antimicrobial protein or peptides.Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria,those produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives.Different classes of LAB bacteriocins have been identified on the basis of biochemal and genetic characterization.These bacteriocins have been reported to inhibit the growth of Listeria monocytogenes,Staphylococcus aureus,Enterococcus faecalis,and Clostridium tyrobutyricum.

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

Result:
Part 1:Determination of bacteriocin activity via agar diffusion test

 

                                  There are inhibition zone between L.Casei and K. peunomia
                                                   Presence of antimicrobial effects

                                          No inbition zone between L. brevis and  S. aereus
                                                      Absence of antimicrobial effects

Strains of LAB	Strains of spoilage/ pathogenic bacteria	Inhibition zone (cm)
Lactobacillus Plantarum	S. aureus	No inhibition zone
	K. pneumonia	(1.40+0.79)/2= 1.095
	P. aeruginosa	(0.70+0.81)/2=0.755
Lactobacillus Brevis	S. aureus	No inhibition zone
	K. pneumonia	(1.40+0)/2=0.700
	P. aeruginosa	(1.10+0)/2=0.550
Lactobacillus Casei	S. aureus	No inhibition zone
	K. pneumonia	(1.00+0.90)/2=0.950
	P. aeruginosa	(0+1.30)/2=0.650

Discussion:
1)Bacteriocins are protein produced by bacteria to inhibit the growth of similar or closely  related bacterial strains.

2)Bacteriocin  inhibit and kill the bacterial by three way,that are:

• Destruction of membrane of the bacterial by forming pores in the membrane.
• Endonucleolytic activity by breakdown the chromosomal DNA as well as RNA.
• Inhibition of protein synthesis but does not lyses the cell.

3)Based on the result,we can seen that there are no inhibition zone present between the Lactobacillus plantarum and S.aureus , Lactobacillus brevis and S.aureus and Lactobacillus Casei and S.aureus.

 

4)There are two reason causes the absent of the inhibition zone between the three LAB strains and S.aureus

     i.The S.aureus that used is unsuccesfully cultured.There are no live S.aureus in the specimen  pathogenic bacterial.

    ii.During the process of adding trypticase soy-yeast extract agar(TSAYE),the temparature might be too high and finally kill the S.aureus .

Part 2:Determination of bacteriocin activity via optical density

Serial dilution of extracellular extract

 Strain of Lab1 : Lactobaciilus plantarum

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain 1: S. aureus	Strain 2: P.aeruginosa	Strain 3: K. pneumonia
0x
2x	0.790	0.836	0.812
10x	0.931	1.125	1.086
50x	0.652	0.495	0.463
100x	0.455	0.462	0.449
Equation	y= -0.004x+0.878	y= -0.005x+0.959	Y= -0.005x+0.925
OD600 of control	0.508	0.129	1.156
50% of OD600	0.508/2=0.254	0.129/2=0.0645	1.156/2=0.578
AU/ml	156	178.9	69.4

 

  
Discussion:
1)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, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. 

2)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)/mWhen y = Z/2, Thus x = (Z/2 - c)/m


3) Generally,the OD value is higher from the 100X dilution. It indicated that the LAB has stronger antimicrobial effect on the pathogenic bacteria compare to a more diluted solution.The  more diluted the extracellular extract, the greater the value of OD<sub>600.

 4)From the result ,the graph can be seen that the optical density of three strains of LAB decrease  when the serial dilution increase.This might caused by using the the distilled water in the process of serial dilution .As  we know that the colour of distilled water is colourless. Lactobacillus plantarum with 100x dilution is approaching colourless,therefore optical density spectrophotometer cannot detect the optical colour density.

5) Peptone is suggested to use because the colour of peptone is similar with the colour of Lactobacillus plantarum culture .Therefore,it will not greatly affect the result.

Conclusion:</sub> 
Bacteriocins produced by Lactic acid bacteria (LAB) as bio preservatives against S.aureus,K. pneumonia and P. aeruginosa. Both acidification and the production of hydrogen peroxide by LAB were ruled out as the source of the inhibition.LAB competed with spoilage microorganisms, such as certain Gram-negative bacteria for nutrients or space with. Moreover, the shelf-life of food products could extend the production of organic acids, hydrogen peroxide, low molecular weight metabolites (such as diacetyl and bacteriocins) due to their inhibiting effect on the growth of spoilage and pathogenic bacteria. 

References: 

http://soongene.net/nonmun/aquaculture287-266.pdf

http://www.biomedcentral.com/1471-2180/10/316/

http://en.wikipedia.org/wiki/Bacteriocin

http://www.academicjournals.org/ajfs/PDF/Pdf2009/Jan/Khalil%20et%20al.pdf

http://people.hofstra.edu/beverly_clendening/Adv_Molecular_Biology/Protocols/Measuring_Optical_Dens.html

 

LAB 5 REPORT BY YI LEE

Name: Choong Yi Lee

Matrix Number: 111359

LAB 5: Determination of Antimicrobial Effects of Microbial Extracts

Introduction:

       Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. 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 lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural 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 ofListeria monocytogens, Staphyloccus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.

Objective:   To determine the antimicrobial effects of extracellular of selected LAB strains.

Results:

Part 1 Determination of bacteriocin activity via agar diffusion test

No inbition zone between L. plantarum and  S. aereus
Absence of antimicrobial effects

There are inhibition zone between L.Casei and K. peunomia
Presence of antimicrobial effects

Strains of LAB	Strains of spoilage/ pathogenic bacteria	Inhibition zone (cm)
Lactobacillus Plantarum	S. aureus	-
	K. pneumonia	(1.40+0.79)/2= 1.095
	P. aeruginosa	(0.70+0.81)/2=0.755
Lactobacillus Brevis	S. aureus	-
	K. pneumonia	(1.40+0) 2=0.70
	P. aeruginosa	(1.10+0) 2= 0.55
Lactobacillus Casei	S. aureus	-
	K. pneumonia	(1.00+0.90) 2=0.95
	P. aeruginosa	(0+1.03)2= 0.515

Discussions:

           1)      An antimicrobial is a substance that kills or inhibits the growth of microorganisms such as       bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body.

2)      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.

3)      The bacteria  is 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.

4)      The lactic acid bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic andphysiological characteristics. These bacteria, usually found in decomposing plants and lactic products, produce lactic acid as the major metabolic end-product of carbohydrate fermentation. This trait has, throughout history, linked LAB with food fermentations, as acidification inhibits the growth of spoilage agents. Proteinaceous bacteriocins are produced by several LAB strains and provide an additional hurdle for spoilage and pathogenic microorganisms.

Part II  Determination of bacteriocin activity via optical density

Serial dilution of extracellular extract

 Strain of Lab1

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain 1: S. aureus	Strain 2: P.aeruginosa	Strain 3: K. pneumonia
0x	-	-	-
2x	0.790	0.836	0.812
10x	0.931	1.125	1.086
50x	0.652	0.495	0.463
100x	0.455	0.462	0.449
Equation	y= -0.004x+0.878	y= -0.005x+0.959	Y= -0.005x+0.925
OD600 of control	0.508	0.129	1.156
50% of OD600	0.254	0.0645	0.578
AU/ml	156	178.9	69.4

Discussions: Part 2 Determination of bacteriocin activity via optical density

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, 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.

Measuring OD600

1) Turn the machine on using the switch in the back of the machine (lower right-hand side as you face the machine). Turn on the monitor screen and printer. Wait for the machine to go through its start up routine.

2) Open the lid on the top of the machine. Choose the large cuvette holder and place it in the holder in front of the light source. The word FRONT should be toward the front of the machine.

3) Click on the "visible light" key to turn the light source on. Quit the diagnostic screen by clicking on the word QUIT in the upper right hand corner. The Main Menu will appear. Choose Single Wavelength mode from the menu that appears. When the next menu appears, make sure that the wavelength used to measure OD is 600. If the menu reads differently, highlight the number and click on it. A keypad will appear, enter 600. Click OK.

4) Place a 500 µl sample of your blank (broth that your bacteria are growing in) in a cuvette. Place the cuvette in the holder.

5) Shut the lid of the machine.

6) Click READ BLANK in the bottom left corner of the screen.

7) After the machine has read the blank, remove the cuvette, replace it with a cuvette containing 500µl of the bacterial culture.

8) Close the lid and click on READ SAMPLES in the upper left hand of the screen.

9) After the machine has read your sample, the data will appear on the screen. Click on Print in the upper right-hand corner of the menu bar to print the data. If you are measuring more than one sample, you do not need to print each time you measure a sample. The machine will collect data for you. You can print when you are finished all of the data collection. You also do not need to blank each time unless your samples are suspended in different solutions.

10) When you are finished reading samples, print your results by clicking on PRINT at the top right of the screen. QUIT the data screen. This will take you back to the main menu. Turn off the light source. You can turn off the machine while the Main Menu screen is active. Turn off the machine, the monitor and the printer.

References:

-http://people.hofstra.edu/beverly_clendening/adv_molecular_biology/Protocols/Measuring_Optical_Dens.html

-http://ag.udel.edu/plsc/labs/sherrierlab/methods_pdf/bacterial%20growth/determining%20bacterial%20concentration%20for%20inoculating%20plants.pdf

-http://en.wikipedia.org/wiki/Lactic_acid_bacteria

-http://en.wikipedia.org/wiki/Agar_diffusion_test

-http://en.wikipedia.org/wiki/Antimicrobial

-http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1622751/

-http://www.webmd.com/vitamins-supplements/ingredientmono-790-LACTOBACILLUS.aspx?activeIngredientId=790&activeIngredientName=LACTOBACILLUS

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Conclusions:

Lactobacillus is a bacteria that can inhibit or kill the growth of microorganism. Lactobacillus is also in some fermented foods like yogurt and in dietary supplements.  lactobacillus can help us break down food, absorb nutrients, and fight off "unfriendly" organisms that might cause diseases.