DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS
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.
Lactic acid bacteria produced organic compound,hydrogen peroxide,propionic acid,diacetyl and bacteriocins.Acetic and propinic acids is produced through heterofermentation pathways,may interact with cell membranes and cause intercellular acidification and protein denaturation.They are antimicrobially effective than lactic acid due to their higher pka value.
Hydrogen peroxide produced in presence of oxygen as a result of the action of flavoprotein oxidase or nicotinamide adenine dinucleotide (NADH) peroxides.The antimicrobacterial effect of hydrogen peroxide result from the oxidation of sulfhydryl group causing denaturating of a number of enzyme and increased membrane permeability.It also acts as a precusor for the production of bacteridial free radical which can damage DNA.
Diacetyl is an aroma compenants produced by citrate fermentation.It inhibit growht of Gram-negative bacteria by reacting with the argining-binding protein,thus affecting the arginine utilization.Diacetyl also act as synergistically with other antimicrobial factors and contributes to combine preservation system.
Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growht of similar related bacteria strains.this bacteriocins are calssified as strain production,common resistance mechanism and mechanism of killing.The bacteriocin in E.coli is called colicins and Staphylococcus warneri are called as warnerin.
OBJECTIVE
To determine the antimicrobial effects of extracellular extracts of selected lactic acid bacteria strains.
RESULT
PART 1 : Determination of bacteriocin activity via agar diffusion test
STRAINS LAB
|
Strains of spoilage / pathogenic
bacteria
|
Inhibitio zone ( cm )
|
L.plantanum
|
S.aureus
|
0.0
|
K.pneumonia
|
0.9
|
|
P.aeruginose
|
0.9
|
|
L.casei
|
S.aureus
|
0.0
|
K.pneumonia
|
0.7
|
|
P.aeruginose
|
0.7
|
|
L.brevis
|
S.aureus
|
0.0
|
K.pneumonia
|
0.6
|
|
P.aeruginose
|
0.7
|
LAB : L.plantanum
 |
| P.aeruginosa |
 |
| K.pneumonia |
 | |||
| S.aureus |
LAB : L.casei
 |
| P.aeruginosa |
 |
| K.pneumonia |
 |
| S.aureus |
 |
| P.aeruginosa |
 |
| K.pneumonia |
 |
| S.aureus |
PART II :Determination of bacteriocin activity via optical density
Dilutions
|
OD600
of spoilage/pathogenic bacteria
|
||
S.aureus
|
K.pneumonia
|
P.aeruginosa
|
|
0X
|
-
|
-
|
-
|
2X
|
0.454
|
0.484
|
0.410
|
10X
|
0.610
|
0.696
|
0.620
|
50X
|
0.386
|
0.571
|
0.312
|
100X
|
0.238
|
0.464
|
0.265
|
Equation
|
y
= 0.013x+0.278
|
y
= 0.113x+0.111
|
y
= -0.031x+0.532
|
OD600
of control
|
0.270
|
0.892
|
0.432
|
50%
of OD600
|
0.135
|
0.446
|
0.216
|
AU/ml
|
-11
|
2.96
|
10.19
|
DISCUSSIONS
PART I
An antibiotic is applied to a well that is cut into the agar. The antibiotic moved from this region of high concentration to the surrounding regions of lower antibiotic concentration. If more material is present in the well, then the zone of diffusion can be larger.
This diffusion was the basis of the agar diffusion . A bacterial suspension is spread onto the surface of the agar.
Then, antibiotic is applied to a number of wells in the plate. There can
be different concentrations of a single antibiotic or a number of
different antibiotics present. Following a time to allow for growth of
the bacteria then agar is examined. If bacterial growth
is right up to the antibiotic containing well, then the bacterial strain
is deemed to be resistant to the antibiotic. There is a clearing
around the antibiotic well, then the bacteria have been adversely
affected by the antibiotic. The size of the inhibition zone can be
measured and related to standards, in order to determine whether the
bacterial strain is sensitive to the antibiotic.
This technique can also be done by placing disks of an absorbent
material that have been soaked with the antibiotic of interest directly
onto the agar surface. The antibiotic will subsequently
This version of agar diffusion is known as the Kirby-Bauer disk-diffusion assay.
The agar diffusion assay allows bacteria to be screened in a routine,
economical and easy way for the detection of resistance. More detailed
analysis to ascertain the nature of the resistance can then follow.
From the result we can see that all the culture has the clear zone,means that there is an inhibition of antimicrobial,but only the S.aureus do not have the inhibition zone.This happen because the culture itself do not strong enough to produce extracellular extract.So that,the supernatant we got do not contain enough extracellular extracts.
There are also factors that can affects zone of inhibition :
1. Pathogen susceptibility
Selection of the antibiotic is based on the type of organism being tested. If the organism is susceptible to the antibiotic, they will not grow near the disk. However, if they are resistant, they will grow right up to the disk.
2. Antibiotic diffusion effects
The rate of diffusion of an antibiotic through the agar is not always same. The rate of diffusion of the antimicrobial through the agar is dependent on the Concentration of antibiotic, Molecular weight of antibiotic, solubility properties of antibiotic, pH and ionization, binding upon agar. Larger molecules will diffuse at a slower rate than lower molecular weight compounds. These factors, in combination, result in each antimicrobial having a unique breakpoint zone size indicating susceptibility to that antimicrobial compound.
3. Agar depth
If the nutrient agar plates are prepared from dehydrated media, the plates must be poured to a depth of four mm. Plates that are too shallow will produce false susceptible results as the antimicrobial compound will diffuse further than it should, creating larger zones of inhibition. Conversely, plates poured to a depth below four mm will result in false resistant results.
4. pH
The nutrient agar medium should be checked through the test. The agar medium should have a pH between 7.2 and 7.4 at room temperature because optimum pH range of most bacteria to grow is pH 6.5- 7.5. Exception Vibrio cholerae is the only human pathogen that grows well above pH 8. If the pH is too acidic, certain drugs (e.g. amino glycosides, quinolone, and macrolides) will appear to loss potency, while other drugs may have excessive activity (tetracyclines, novobiocin, methioillin) and thus result in a smaller or larger zone of inhibition. If the pH is besides high, the opposite effects can be expected.
5. Size of the inoculated organism:
The size of the inoculated organism must also be standardized. The reasons are because if the size of the inoculum is too small, the zone of inhibition will be larger than what it is supposed to be and if the inoculum are also large, the zone of inhibition will be smaller.
6. Presence of other metals:
Excessive thymidine or thymine can reverse the inhibitory effects of sulfonamides and trimethoprim resulting in smaller and fewer distinct zones of inhibition, or no zones at all. The incorrect concentration of divalent cations (calcium and magnesium) will affect the results of aminoglycoside and tetracycline tests against Pseudomonas aeruginosa. Excess cation concentration will result in reduced zone sizes, and low concentration will increase zone sizes. Excess calcium will increase the zone size of P. aeruginosa against daptomycin. Excess zinc ions may reduce the zone size of carbapenems against P. aeruginosa. Zone of inhibition also affected by the Concentration of bacteria spread onto agar plate, Drug antagonists, incubation temperature, incubation time, size of the plates, proper spacing of the disks, reading of the zone, etc.
Selection of the antibiotic is based on the type of organism being tested. If the organism is susceptible to the antibiotic, they will not grow near the disk. However, if they are resistant, they will grow right up to the disk.
2. Antibiotic diffusion effects
The rate of diffusion of an antibiotic through the agar is not always same. The rate of diffusion of the antimicrobial through the agar is dependent on the Concentration of antibiotic, Molecular weight of antibiotic, solubility properties of antibiotic, pH and ionization, binding upon agar. Larger molecules will diffuse at a slower rate than lower molecular weight compounds. These factors, in combination, result in each antimicrobial having a unique breakpoint zone size indicating susceptibility to that antimicrobial compound.
3. Agar depth
If the nutrient agar plates are prepared from dehydrated media, the plates must be poured to a depth of four mm. Plates that are too shallow will produce false susceptible results as the antimicrobial compound will diffuse further than it should, creating larger zones of inhibition. Conversely, plates poured to a depth below four mm will result in false resistant results.
4. pH
The nutrient agar medium should be checked through the test. The agar medium should have a pH between 7.2 and 7.4 at room temperature because optimum pH range of most bacteria to grow is pH 6.5- 7.5. Exception Vibrio cholerae is the only human pathogen that grows well above pH 8. If the pH is too acidic, certain drugs (e.g. amino glycosides, quinolone, and macrolides) will appear to loss potency, while other drugs may have excessive activity (tetracyclines, novobiocin, methioillin) and thus result in a smaller or larger zone of inhibition. If the pH is besides high, the opposite effects can be expected.
5. Size of the inoculated organism:
The size of the inoculated organism must also be standardized. The reasons are because if the size of the inoculum is too small, the zone of inhibition will be larger than what it is supposed to be and if the inoculum are also large, the zone of inhibition will be smaller.
6. Presence of other metals:
Excessive thymidine or thymine can reverse the inhibitory effects of sulfonamides and trimethoprim resulting in smaller and fewer distinct zones of inhibition, or no zones at all. The incorrect concentration of divalent cations (calcium and magnesium) will affect the results of aminoglycoside and tetracycline tests against Pseudomonas aeruginosa. Excess cation concentration will result in reduced zone sizes, and low concentration will increase zone sizes. Excess calcium will increase the zone size of P. aeruginosa against daptomycin. Excess zinc ions may reduce the zone size of carbapenems against P. aeruginosa. Zone of inhibition also affected by the Concentration of bacteria spread onto agar plate, Drug antagonists, incubation temperature, incubation time, size of the plates, proper spacing of the disks, reading of the zone, etc.
PART II
Spectrophotometer is is a quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the common term electromagnetic spectroscopy which deals with visible light near ultraviolet and near infra-red. It does not cover the time resolved spectroscopic techniques which means that "anything that allows to measure temporal dynamics and kinetics of photo physical processes". Spectrophotometry involves the use of spectrophotometer.
From the experiment,result for Strain 1 and Strain 2 get as we aspectted.The optical density increases when the serial dilution increases.However,we do not get the right result for Strain 3 because when the serial dilution increases,the optical density decreases.There must be wrong technique uses in the serial dilution of distilled water.This is because when we do the dilution ,the concentration of pathogenic bacteria is getting low so do the color density.The color density is not the right color that we should get for this type of culture.When there is more dilution of distilled water,the color density of the culture become clear.Thus,we need to replace the distilled water with peptone because the color is similar to the Strain 3.
CONCLUSION
Lactic acid bacteria are able to produce a large variety of compounds which
give fermented foods their characteristics flavor and color,and also impart improve safety and rheology to the foods.The production of antimicrobial compounds and extracellular polysaccarides by LAB strains obtained from dairy industry has been investigated in order to find the LAB strains with potential applications in food.
REFERENCES
http://en.wikipedia.org/wiki/Bacteriocin
http://www.doria.fi/bitstream/handle/10024/3085/antimicr.pdf?sequence=2
http://en.wikipedia.org/wiki/Agar_diffusion_test
http://www.innovateus.net/science/what-spectrophotometer
http://www.innovateus.net/science/what-spectrophotometer
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