LAB 5 :
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 protozoa. Antimicrobial drugs either
kill microbes or prevent the growth of microbes . Disinfectants are antimicrobial substances used on non-living
objects or outside the body.
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 heavy metal.
The lactic acid
bacteria (LAB) comprise a of Gram-positive, low-GC,
acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are
associated by their common metabolic and physiological 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.
OBJECTIVE
To determine the antimicrobial
effect of extracellular extracts of selected LAB strain.
RESULTS
Part 1 : Determination of
bacteriocin activity via agar diffusion test.
i. L.plantarum
ii. L.brevis
iii. L.casei
TABLE 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
|
0.00
|
|
L.brevis
|
S.aureus
|
0.00
|
K.pneumoniae
|
0.70
|
|
P.aeruginosa
|
0.80
|
|
L.casei
|
S.aureus
|
0.00
|
K.pneumoniae
|
1.00
|
|
P.aeruginosa
|
0.65
|
Part 2 : Determination of bacteriocin activity via optical density.
Strain of lab : L.plantarum
DILUTION
|
OD600 of spoilage / pathogenic bacteria
|
||
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 2 :
STRAIN 3 : K.pneumoniae
DISCUSSION
Part 1 : Determination of bacteriocin activity via
agar diffusion test.
The concentration
of the antimicrobial will be higher next to the disk, and will decrease
gradually as distance from the disk increases. If the antimicrobial is
effective against bacteria at a certain concentration, no colonies will grow
wherever the concentration in the agar is greater than or equal to that
effective concentration. This region is called 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 (zone of inhibition) around the filter disk, the more effective the antimicrobial.
In this experiment, we use 3 types of bacteriocin ; L.plantarum, L.brevis, and L.casei.
We can see that from the result, for L.plantarum, the
inhibition zone in K.pneumoniae is
largest. Which means, the effectiveness of L.plantarum
towards this pathogen is high. While for S.aureus
and P.aeruginosa, the
effectiveness of this bacteriocin is lower than in K.pneumoniae as the zone of inhibition are small in diameter.
Secondly , for bacteriocin L.brevis, the diameter of inhibition
zone in P.aeruginosa is the largest
compared to S.aureus and K.pneumoniae. This indicates that the
effectiveness of L.brevis to inhibit the growth of pathogen
is the highest toward P.aeruginosa.
Thirdly , for bacteriocin L.casei, it has the highest effectiveness of inhibition towards K.pneumoniae as the zone of inhibition
in K.pneumoniae agar plate is the
largest diameter. This bacteriocin has zero level of inhibition towards
pathogen S.aureus , same goes with
bacteriocin L.brevis.
Growth inhibition techniques for quantitative determination of
bacteriocins also relies on the sensitivity of the applied indicator
microorganism to the bacteriocin to which is exposed.
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.
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.
Although the agar diffusion method is the most widely used method in routine
measurements of bacteriocin activity, optical density offers a simpler, faster
and more reliable alternative since diffusion related problems are eliminated,
the degree of human intervention and judgment is low, and very low bacteriocin
concentrations can be quantified.
REFERENCES
en.wikipedia.org/wiki/Bacteriocin
fst.sagepub.com/content/7/4/281.abstract
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