Diffusion of Gases Through the Respiratory Membrane

DISUSUN OLEH:
BAGUS MUHAMMAD IHSAN
130120160003

ILMU KEDOKTERAN DASAR

FAKULTAS KEDOKTERAN
UNIVERSITAS PADJADJARAN
BANDUNG
2016

ANTI MICROBIAL EFFECT OF BACTERIOCINS Lactobacillus acidophilus ON THE

GROWTH OF Staphylococcus aureus

MATERIALS AND METHODS

Microbial

consist

of

0,02%, MnSO4 0,005%) nutrient agar and

Lactobacillus

acidophilus and Staphylococcus aureus taken

from original isolate which is obtained from
Bacterial Laboratory of Biofarma.
Morphological
identification.
Bacterial

isolates

were

identified

by

broth brain heart infussion (BHI) which is used

as growth media for Lactic Acid Bacteria
(LAB) that produced

bacteriocins, media

Mueller hinton agar (MHA) is used as a

medium for tested the activity of bacteriocins.
Reidentified

and

Rejuvenated

of

microscopic observation based the shape of the

bacteria. Before used, we reidentified and

bacteria using Gram staining.

Catalase test. One loop of isolate was

rejuvenated the Lactobacillus acidophilus.

smeared. It put on object glass that have been

cleaned by alcohol. Drops of 3% H2O2 solution
were added. The formation of gas bubbles
indicate that isolate were catalase positif
(Gawad et al, 2010).
Medium for bacterial growth are man

Lactic acid bacteria Lactobacillus acidophillus

rejuvenated in MRS broth with yeast extract
for several times until the bacteria grew well.
This process was also done on bacteria
Staphylococcus aureus in nutrient broth.
Single isolates from solution identified

rogosa, and sharpe (MRS) broth and agar, CM

by Gram stain, catalase test and oxidase test.
sucrose (Sucrose 1%, yeast exstract 1%, pepton
Original isolates are Gram (+), catalase (-) and
0,45%, KH2PO4 2,84%, NaCl 0,2%, MgSO4
oxidase (-) was a LAB isolates. Isolates stored

at 4 C in CM Sucrose slant agar. Isolates were

enzymes effect were conducted as 200 L of

stored at 4 C refreshed with CM sucrose

the supernatant bacteriocins dissolved in 20 L

medium when it will be used.

of an enzyme solution (enzyme solution

Production of Bacteriocin. Production

of bacteriocins refers from Ogunbanwo et al.
(2003). Isolates which selected were grown on
CM sucrose at 35 C for 48 hours. Culture
cells that formed were centrifuged (8,000 rpm,
4 C for 15 minutes) to obtain the supernatant.
The supernatant that free of cell were
regulated up to pH 6 by added 1 M NaOH (to
eliminated the effect of organic acids), and then
filtered with a cellulose acetate filter 0.2 m.
Furthermore, supernatant was heated at 80 C
for 10 minutes to removed the proteolytic
activity and hydrogen peroxide. The last

Bacteriocin Sensitivity Test against

proteolytic enzymes. To confirmed that the
supernatant was bacteriocins, test activity of the
enzymes.

Supernatant

phosphate buffer pH 7), and then incubated at

37 C for 2 hours. Supernatant sensitive to
proteolytic enzymes is the supernatant without
clear zone / halo firmly around sinks.
Bacteriocin

Test.

Bacteriocin

test

activity performed using the well diffusion

method (Ogunbanwo et al., 2003; Udhayashree
et al., 2012). 100 mL of indicator bacteria
inoculated on MHA, then made 6 mm diameter
wells on agar. In each of the wells included
10L bacteriocins. Then, incubated at 35 C
for 24 hours.
Bacteriocin activity shown by the clear

supernatant was bacteriocins.

proteolytic

prepared by dissolve the enzyme in NaOH or

loss

of

activity after the proteolytic enzyme test was an

actual bacteriocins. Proteolytic enzyme used
was papain and proteinase K. Assessment of the

zone / halo firmly around sinks. Bacteriocin

activity is expressed as arbitrary units (AU) per
mL. One AU is expressed as broad zones of
inhibition per unit volume of samples tested
(mm2/ml)

bacteriocins

(Usmiati

Marwati,2007).
Bacteriosin activities (mm2 / ml) = AU / mL
= Lz Ls

&

V
Lz = wide clear zone (mm2)

The sample and control tube incubated

at 37 C for 60 minutes, then tested for

Ls = wide of sink (mm2)

bacteriocin activity using diffusion method
V = sample volume (mL)
agar. Bacteriocin activity expressed in AU / ml.
The

temperature

resistance

test.

Bacteriocin characterization. In 5 ml
Bacteriocins taken 200 l then tested for
bacteriocins in tubes, set each tube at pH 2, 4,
resistance to 50C for 20 minutes, 100oC for 20
6, 8, and 10 using NaOH or HCl solution. After
minutes

and

121oC

for

15

minutes

incubation for 4 hours at room temperature,

(Osmanagaoglu et al., 1998; Mandal et al.,
bacteriocin activity was tested using the
2008).
diffusion method agar.

Bacteriocin activity
Bacteriocins stability during storage.

expressed in AU / ml (Usmiati & Marwati,

Stability

test

was

done

by

stored

the

2007).
bacteriocins at -20, 4, and 37 C for two
Effect of surfactant. Surfactants used
months. Furthermore, the bacteriosin activity
were triton X-100, Tween 20, tween 80, sodium
test by the method of diffuse agar (Nugroho &
dodecyl sulfate / SDS, EDTA, urea and NRahayu, 2003).
Laurylsarcosine. Surfactants were added to
each solution bacteriocins at a concentration of
0.1ml or 0.01 g surfactant per ml bacteriocins.
Note :
Bacteriocins solution without the surfactant
Instruction

for

the

author

used as a control.
Microbiology Indonesia Journal

reference

to