Lab 4271

LAB 5: DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS

INTODUCTION

Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acid, 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 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 biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Listeria monocytogenes, Staphylococcus aures, Enterococcus faecalis and Clostridium tyrobutyricum.

Two methods can be used. First determination of bacterion activity using agar diffusion test and using optical density.

Part 1: Determination of bacterion activity via agar diffusion test

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.

The bacteria in question 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. In microbiology, minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. A lower MIC is an indication of a better antimicrobial agent. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism.

Part 2: Determination of bacterion activity via optical density.
A spectrophotometer is an optical device that can determine the concentration of a compound or particles in a solution or suspension.

Light of a pre-selected wavelength is shone through a chamber that houses the sample. The sample particles, bacteria for example, will absorb some of the light. The amount of light that is absorbed increases with increasing numbers of bacteria in a predictable way. The relationship between absorbance and the number of absorbing sample molecules is expressed mathematically as the Beer-Lambert Law.The percent of light that has been absorbed can be determined and, by comparing this absorption to a graph of the absorption of known numbers of bacteria, the concentration of bacteria in the suspension can be computed. In a microbiology laboratory, such measurements are routinely used in bacterial growth studies, to determine the number of bacteria growing in a culture at certain times based on the absorbance of the suspension. A standard curve can be constructed that relates the various measured optical densities to the resulting number of living bacteria, as determined by the number of bacteria from a defined portion of the suspensions that grows on agar medium.

OBJECTIVE

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

RESULTS
PART 1: Determination of bacterion activity via agar diffusion test

Calculation

DATA SHEET

PART 1 : DETERMINATION OF BACTERIOCIN ACTIVITY VIA AGAR DIFFUSION TEST

STRAINS OF LAB	STRAINS OF SPOILAGE / PATHOGENIC BACTERIA	INHIBITION ZONE (cm)
L.plantarum	S.aureus	0.60
	K.pneumoniae	1.15
	P.aeruginosa	-
L.brevis	S.aureus	-
	K.pneumoniae	0.70
	P.aeruginosa	0.80
L.casei	S.aureus	-
	K.pneumoniae	1.00
	P.aeruginosa	0.65

L.plantarum

L.brevis

L.casei

Part 2: Determination of bacterion activity via optical density.

Serial dilution of extracellular extract

Strain of lab : L.plantarum

DILUTION	OD₆₀₀of spoilage / pathogenic bacteria
DILUTION	STRAIN 1: P.aeruginosa	STRAIN 2: S.aureus	STRAIN 3: K.pneumoniae
0x	-	-	-
2x	1.025	0.787	0.871
10x	0.733	0.772	0.595
50x	0.755	0.560	0.506
100x	0.260	0.321	0.237
EQUATION	Y= -0.2273X + 1.4888	Y= -0.161X + 1.1735	Y= -0.1991X + 1.2491
POSITIVE CONTROL(Z)	0.432	0.270	0.829
50% of POSITIVE CONTROL (Z/2)	0.216	0.135	0.4215
AU/mL	5.5996	6.4503	4.1919

STRAIN 1: P.aeruginosa

STRAIN 2: S.aureus

STRAIN 3: K.pneumoniae

DISCUSSION

PART 1: Determination of bacterion activity via agar diffusion test

In this experiment, we need to determine the antimicrobial effects of extracellular extracts of selected LAB strains. 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).The principle of this method is dependent upon the inhibition of reproduction of a microorganism on the surface of a solid medium by an antimicrobial agent which diffuses into the medium from a filter paper disc. Thus, for an organism which is truly sensitive (susceptible) to an antimicrobial agent The principle of this method is dependent upon the inhibition of reproduction of a microorganism on the surface of a solid medium by an antimicrobial agent which diffuses into the medium from a filter paper disc. Thus, for an organism which is truly sensitive (susceptible) to an antimicrobial agent. Accurate measurement of zone diameter is necessary to properly interpret this test.

Lactobacilli is the inhibitor that inhibit most of all the microbial activity of Gram positive bacteria compare to the Gram negative bacteria. In this experiment, S.aureus is the type of Gram positive bacteria, K.pneumoniae and P.aeruginosa the type of Gram negative. Based on the result obtain, L.plantarum inhibit the activity of S.aureus and the inhibition zone is 0.6cm. There were an error occured during the procedure because S.aureus should gave the highest result compared to the two patogen. The depth of the paper disk are not deep enough and thus gave the error in our result.

Part 2: Determination of bacterion activity via optical density.

In part 2, we need to determine the bacterial activity via optical density(spectrophotometer). Upon the incubation process, the measurement of the optical density of the spoilage or pathogenic bacteria is taken at 600nm. From the experiment, the arbitrary unit(AU/mL) or the dilution factor of the extracellular extract that inhibit 50% of the spoilage is determined. Several dilution has been done and the 2x dilution gaves the highest reading for the three of the species. After the calculation, it have been determined that S.aureus gave the higest value compare the two. The gram positive gave the higher value compare to the gram negative.

CONCLUSION

Agar diffusion test is fast and inexpensive relative to other laboratory tests for antimicrobial activity. In addition, it requires media, reagents, equipment and supplies that are readily accessible to most clinical laboratories. Agar diffusion test is especially well suited for determining the ability of water-soluble antimicrobials to inhibit the growth of microorganisms. A number of samples can be screened for antimicrobial properties quickly using this test method.

The spectrophotometric technique enables results to be available in 24 h, for various antimicrobial–microorganism combinations, easily and conveniently and with results that can be replicated. For every antimicrobial agent–microorganism association and for every antimicrobial concentration tested, the results obtained are not significantly different from those obtained with the viable count technique.

REFERENCES

http://www.enotes.com/spectrophotometer-reference/spectrophotometer

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

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

http://jac.oxfordjournals.org/content/47/4/391.full

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

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

Introduction:

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.

The history of antimicrobials begins with the observations of Pasteur and Joubert, who discovered that one type of bacteria could prevent the growth of another. They did not know at that time that the reason one bacterium failed to grow was that the other bacterium was producing an antibiotic. Technically, antibiotics are only those substances that are produced by one microorganism that kill, or prevent the growth, of another microorganism. Of course, in today's common usage, the term antibiotic is used to refer to almost any drug that attempts to rid your body of a bacterial infection. Antimicrobials include not just antibiotics, but synthetically formed compounds as well.

The discovery of antimicrobials like penicillin and tetracycline paved the way for better health for millions around the world. Before penicillin became a viable medical treatment in the early 1940s, no true cure for gonorrhea, strep throat, or pneumonia existed. Patients with infected wounds often had to have a wounded limb removed, or face death from infection. Now, most of these infections can be cured easily with a short course of antimicrobials.

However, with the development of antimicrobials, microorganisms have adapted and become resistant to previous antimicrobial agents. The old antimicrobial technology was based either on poisons or heavy metals, which may not have killed the microbe completely, allowing the microbe to survive, change, and become resistant to the poisons and/or heavy metals.

Antimicrobial nanotechnology is a recent addition to the fight against disease causing organisms, replacing heavy metals and toxins and may some day be a viable alternative.

Infections that are acquired during a hospital visit are called "hospital acquired infections" or nosocomial infections. Similarly, when the infectious disease is picked up in the non-hospital setting it is considered "community acquired".

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 produce antimicrobial agents, such as bacteriocins, is on the rise, since nowadys 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 of Listeria monocyotogenes,Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.

Objectives:

To determine the antimicrobial effect of extracellular extracts of selected LAB strain.

Results:

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

L. plantarum
K. pneumoniae

L. plantarum ;
S. aureus

L. plantarum ;
P. aeruginosa

L. brevis ;
K. pneumoniae

L. brevis ;
S. aureus

L. brevis ;
P. aeruginosa

L. casei ;
K. pneumoniae

L. casei ;
S. aureus

L. casei ;
P. aeruginosa

Strains of LAB	Strains of spoilage / Pathogenic Bacteria	Inhibition Zone (cm)
L.plantarum	S.aureus	0.60
	K.pneumoniae	1.15
	P.aeruginosa	-
L.brevis	S.aureus	-
	K.pneumoniae	0.70
	P.aeruginosa	0.80
L.casei	S.aureus	-
	K.pneumoniae	1.00
	P.aeruginosa	0.65

Part 2 : Determination of bacterion activity via optical density.

Serial dilution of extracellular extract
Strain of lab : L.plantarum

Dilution	OD₆₀₀of spoilage / pathogenic bacteria
Dilution	Strain 1: P.aeruginosa	Strain 2: S.aureus	Strain 3: K.pneumoniae
0x	-	-	-
2x	1.025	0.787	0.871
10x	0.733	0.722	0.595
50x	0.755	0.560	0.506
100x	0.260	0.321	0.237
Equation	Y= -0.2273X + 1.4888	Y= -0.156X + 1.1435	Y= -0.1991X + 1.2491
Positive Control (Z)	0.432	0.270	0.829
50% of Positive Control (Z/2)	0.216	0.135	0.4215
AU/mL	5.5996	6.4503	4.1919

Discussion:

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

Using sterile inoculation loop, a loop full of direct Leuconostoc mesenteroides culture was touched over the swabbed plate and a small smear was made. A drop of the soft agar was dropped into the well to seal the bottom. The test organism Salmonella typhi, Escherichia coli, and Shigella flexinerrae were swabbed on the respective plates. The bacteriocin was extracted and its antagonistic activity was studied against the indicator organisms by well diffusion method

Bacteriocin was extracted by cell free supernatant method and the crude supernatant was determined for its antagonistic activity. A well diffusion method was performed. In the activity it was observed that the bacteriocin produced by L.mesenteroides was effective against Salmonella typhi and Escherichia coli. But there was no effect against Shigella flexinerrae. The reviews say that the bacteriocins produced by L.mesenteroides are active against Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Bacillus cereus, L.monocytogenes has been considered as the major food borne pathogen and most activities were against them in food industries. Now these studies reveal the scope for bacteriocins, not only as preservatives but also as an antibiotic for many diseases and infections. Bacteriocin activity was determined in an agar well diffusion assay. To test the ability of the polyclonal antiserum to neutralize bacteriocin activity, serial dilutions of bacteriocin were mixed with an equal volume of undiluted antiserum in each well prior to adding the overlay. Preimmune serum and sterile deionized water were mixed with bacteriocin in the control wells. All tests were run in duplicate. This is an area around a paper disk or colony of bacteria(LAB) or mold where no other organisms are growing. If you are testing antibiotic sensitivity for example, you can impregnate paper disks with antibiotic and then put them on an agar plate of growing bacteria. The antibiotic then diffuses into the agar away from the disk. If the bacteria are sensitive to the antibiotic, they will not grow near the disk. The size of the zone is proportional to how sensitive the organism is. If the organism is resistant to the antibiotic, it will grow right up to the disk.

Part 2 : Determination of bacterion 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.

A spectrometer (spectrophotometer, spectrograph or spectroscope) is an instrument used to measure properties of lightover a specific portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify materials. The variable measured is most often the light's intensity but could also, for instance, be the polarization state. The independent variable is usually the wavelength of the light or a unit directly proportional to the photon energy, such as wavenumber or electron volts, which has a reciprocal relationship to wavelength. A spectrometer is used in spectroscopy for producing spectral lines and measuring their wavelengths and intensities. Spectrometer is a term that is applied to instruments that operate over a very wide range of wavelengths, from gamma rays and X-rays into the far infrared. If the instrument is designed to measure the spectrum in absolute units rather than relative units, then it is typically called a spectrophotometer. The majority of spectrophotomers are used in spectral regions near the visible spectrum.

In general, any particular instrument will operate over a small portion of this total range because of the different techniques used to measure different portions of the spectrum. Below optical frequencies (that is, at microwave and radio frequencies), the spectrum analyzer is a closely related electronic device.

Conclusion:

LAB can produce a useful bacterioncin such as L.plantarum, L.brevis, and L.casei that can effectively inhibit the growth of several pathogen such as K.pneumoniae, S.aureus and P.aeruginosa.

Bacteriocins from lactic acid bacteria are of importance in bioconservation of various foods. Moreover, the use of more than one LAB bacteriocin as a combination of biopreservative may have major applications in improving food safety. In the present study, the inhibitory effect of the cell-free filtrates of each of the 20 isolates was evaluated. Antimicrobial activity was observed for 6 isolates, and only against Gram positive bacteria. The biochemical nature of the antibacterial molecule produced by S. thermophilus T2 was studied in both the cell-free supernatant and the chloroform extract.

References: