Antifungal Effect Of Lactobacilli lactis On Candida albicans Yeast
Shirin Khosravinejat and Setareh Zareh
Department of Biological Sciences
Saddleback College
Mission Viejo, CA, 92692
Candida albicans has a huge impact on human growth factors, yet Lactobacillus is a
bacterium that can decrease candida and the infections it causes on the immune system.
With the overuse of antibiotics, candida has grown to resist antibiotics and grow and affect
the immune system, yet lactobacilli still has an impact of candida’s growth. It was predicted
that as lactobacillus inhibits the growth of Candida albicans. The experiments were done
by serial diluting of both samples with MRS broth, which were grown in a mixed culture
and compare them to a pure culture growth of candida in thioglycollate broth, and data
were by hemocytometer throughout 24, 48, and 72, 96hrs of incubation. Growth rate of
Candida albicans in pure culture was 5.926 × 107 ± 6.09 × 107 and the growth rate of
Candida albicans in mixed culture (with lactobacillus) was 4.36 × 107 ± 1.68 × 106, p =
0.0287. As the lactobacillus acidified the environment, the growth of Candida albicans
started to decrease during the 24hrs throughout 96hrs of incubation. So it was proved that
lactobacillus inhibited the growth of Candida albicans due to the production of lactic acid.
Introduction
Our study is looking at how Lactobacilli will decrease
the growth of Candida albicans. Candida has a huge
impact on human population and can cause infections
on human growth rate factors. Candida and lactobacilli
are both highly prevalent in the human population. A
balance of protective bacteria in the human body
normally controls Candida bacteria growth.
Lactobacillus is a bacteria strain responsible for
protection against pathogenic microorganisms.
Lactobacillus protects the body by producing lactic
acid and other antimicrobial products reducing the
pathogenic bacteria growth rate (Wagner, et al., 2012).
The overuse of antibiotics and other
immunosuppressive agents has altered the immune
system, and caused the increase of Candida infections.
Lactobacilli levels decrease with the use of antibiotics
that are introduced to human bodies. Researchers have
conducted experiments to see how the relation between
lactobacilli bacteria and candida, and how the presence
Materials and Methods
Research study took place at Saddleback
College Biology Laboratory (Mission Viejo,
California) between 6 November 2013 and 20
November 2013. Enriched Thioglycollate broth (pH≥
6.5±0.2) was selected for pure culture of Candida
albicans as a control. One pack of Thioglycollate broth
consists of 6 broth tubes (5 ml each) were provided
from microbiology department of Mission hospital
laboratory in Mission Viejo, California. MRS broth
of antibiotics effect the levels of lactobacilli in healthy
humans. When both bacteria are present together
lactobacilli shows significantly higher levels and
candida growth rate is decreased as opposed to when
the bacteria’s are separately grown suggesting an
increased response from the protective lactobacilli
bacteria in the presence of the pathogenic candida
bacteria (Okkers, et al., 1999). The increased response
of the lactobacilli bacteria, and its lactic acid
production which creates and unfriendly environment
for bacteria growth, suppresses the growth of the
candida bacteria when compared to the growth of
candida separately. The function of the lactobacilli
bacteria and the rate of response growth is decreased
when antibiotics are introduced. Antibiotics role is to
decrease the rate of all bacteria growth as such both
lactobacilli and candida levels decrease with antibiotics
are present.
was selected for mixed culture of both C. albicans and
Lactobacillus lactis and all ingredients were provided
by college laboratory. One hundred ml MRS broth was
made in the laboratory and final pH of broth was
measured by pH meter and recorded as (pH ≥ 6.5
±0.2). MRS broth were poured into 15 glass tubes (5
ml each) and autoclaved for 15 minutes at 121°C.
Strains of Candida albicans provided by
Saddleback college biology department were cultured
in five individual tubes of thioglycollate broth with
volume of 5 ml separately as a control and mixed with
Lactobacillus lactis in 5 tubes of MRS broth with 5 ml
1
volume and incubated 24 hours at 37°C. Grown
colonies in pure and mixed cultures were observed
under light microscope with 400× magnification to
distinguish between Lactobacillus and Candida strains.
Ability of the pro biotic bacteria to inhibit growth of C.
albicans was evaluated using a culture overlay
technique in which pure and mixed culture were
separately diluted to 107 µl in five sets of samples into
seven micro centrifuge tubes labeled from 101 to 107.
Using aseptic technique and 1000 µl micropipette
(P1000) with volume set to 900 µl, 900µl of MRS
broth were pipette into each 35 micro centrifuge tubes
for 5 sets of mixed culture (n=5). Using 200 µl
micropipette (P200) with volume set to 100 µl, 100 µl
of first mixed culture broth of Lactobacillus and
Candida were added to 101 micro centrifuge tubes for
set number one and were mixed by vortex (VWR
Scientific Products) mixer accordingly. One hundred µl
of tube 101 were removed and transferred into 102
micro centrifuge tubes and serial dilution were
completed to 107 then procedure was repeated for four
remaining sets and total number of 35 micro centrifuge
tubes consist of diluted mixed Lactobacillus and C.
albicans obtained. Five sets of thioglycollate broth
consist of pure C. albicans were also Diluted using
same technique and total number of 35 diluted tubes of
pure C. albicans obtained. All 70 pure and mixed
diluted micro centrifuge tubes then incubated for 24
hours at 37°C. From serial diluted micro centrifuge
tubes, five sets of tubes with suspension 107 were
selected for the pure and mixed cultures to be counted
using hemocytometer at 24, 48, 72 and 96 hours of
incubation. To prepare the hemocytometer, the mirrorlike polished surface was cleaned with lens paper and
95% ethanol and a coverslip placed on the grids prior
to adding cell suspension. Cell suspension was mixed
10 sec by vortex and 20 µl of sample drawn and
introduced into one of the V-shaped grids under
coverslip. The counting chamber was then placed
under microscope with magnification set to 100× and
five squares out of nine large squares (four outer
squares and one central square) were selected for
count. Cells that overlapped a ruling counted in if they
overlapped the top or right ruling and out if they
overlapped bottom or left ruling. Candida cells counted
for 24, 48, 72 and 96 hours of incubation and data
recorded accordingly. Knowing that each large square
of hemocytometer represented a total volume of 0.1
mm³ or 10-4 cm³. Since 1cm³ is equivalent to 1 ml, the
total number of cell per 1ml was determined using the
following calculations:
Dilution Factor= Volume of sample = 1000𝜇l
= 10
Volume of cells
100 𝜇l
Total number of cells/ml = total number of cells
counted in all × Dilution factor × 104 cells/cm³
5
squares
number of squares
Total numbers of cells per 5ml of original samples for
each five original tubes of pure and mixed culture were
also calculated using following formula:
Total number of cells/5ml sample = Total number of
cells per 1ml × 5
Total number of cells per 5 ml of five original samples
were added and divided by five to get the average of
five initial samples for both pure and mixed cultures. A
pH control test was run on mixed and pure broth to
confirm the decrease in pH by production of lactic
acid, which inhibited growth of C. albicans. Calculated
data for Candida pure culture and mixed culture at 24,
48, 72 and 96 hours were analyzed on MS Excel
(Microsoft Corporation Redmond Washington) using
unpaired t-test for growth of candida in pure and mixed
cultures and ANOVA and Bonferroni corrections for
difference in growth at 24, 48, 72and 96 hours of
incubation.
Results
Candida albicans cells counted in thioglycollate broth
for five sets of pure samples and in MRS broth for five
sets of mixed samples at 24, 48, 72 and 96 hours of
incubation and data recorded
are shown in table1. Total numbers of cells per 1ml
calculated for five tubes in both pure and mixed culture
and the results are shown in table2. Total numbers of
cells/5ml of original samples were also calculated and
average number of C.albicans cells obtained from five
sets of tubes for both cultures and shown in table3 and
4. Data analyzed using one tailed unpaired t-test and
mean graphs of typical growth are shown in figure1.
There was a consistent marked decrease in Candida
when grown with lactobacilli. All five tubes of mixed
culture of Candida and Lactobacillus showed this
decrease, however not in a same ratio. P value =
0.00235276 supported the hypothesis of the study that
Lactobacilli cause a significant decrease in growth of
Candida albicans.
Hours
of
incubati
on
# Of Candida counted in 5 squares in pure
micro tube with suspention10⁷
Tube2
24
Tube
1
530
Tube
4
1340
Tube 5
347
Tube
3
940
48
477
265
711
955
789
1012
2
72
397
228
681
890
533
96
273
128
550
761
432
in 5 squares in
24
# of Candida Counted
mixed micro tube 10⁷
Tube
Tube
Tube
1
2
3
102
53
167
Tube
4
425
Tube
5
188
48
54
27
77
120
102
72
40
11
40
65
22
96
28
7
25
21
15
4
10
5
10
5
1.0
0E
+0
4
1.0
0E
+0
4
8.00
E+0
5
2.20
E+0
5
8.00
E+0
5
1.30
E+0
6
4.40
E+0
5
5.60
E+0
5
1.40
E+0
5
5.00
E+0
5
4.20
E+0
5
3.00
E+0
5
Table 2. Total number of Candida cells calculated per
1ml at 24, 48, 72, 96 hours of incubation.
Total # of Candida per 5 ml of sample
Table 1. Number of Candida cells counted in all five
squares of hemocytometer at 24, 48, 72, 96 hours of
incubation.
Dilu
tion
fact
or
10
#
Of
squ
ares
5
10
5
10
5
10
5
Dilu
tion
fact
or
# of
squ
ares
10
5
10
5
Sus
pen
sio
n
1.0
0E
+0
4
1.0
0E
+0
4
1.0
0E
+0
4
1.0
0E
+0
4
Sus
pen
sio
n
1.0
0E
+0
4
1.0
0E
+0
Total # of Candida cells per ml
Tub
e1
1.06
E+0
7
Tub
e2
6.94
E+0
6
Tub
e3
1.88
E+0
7
Tub
e4
2.68
E+0
7
Tub
e5
2.02
E+0
7
9.54
E+0
6
5.30
E+0
6
1.42
E+0
7
1.91
E+0
7
1.58
E+0
7
7.94
E+0
6
4.56
E+0
6
1.36
E+0
7
1.78
E+0
7
1.07
E+0
7
5.46
E+0
6
2.56
E+0
6
1.10
E+0
7
1.52
E+0
7
8.64
E+0
6
Total # of Candida cells per ml
Tube 1
Tube 2
Tube3
Tube 4
Tube 5
5.30E+0
7
4.77E+0
7
3.97E+0
7
2.73E+0
7
3.47E+0
7
2.65E+0
7
2.28E+0
7
1.28E+0
7
9.40E+0
7
7.11E+0
7
6.81E+0
7
5.50E+0
7
1.34E+0
8
9.55E+0
7
8.90E+0
7
7.61E+0
7
1.01E+
08
7.89E+
07
5.33E+
07
4.32E+
07
Total # of Candida per 5 ml of sample
Tube 1
Tube 2
Tube3
Tube 4
Tube 5
1.02E+0
7
5.40E+0
6
4.00E+0
6
2.80E+0
6
5.30E+0
6
2.70E+0
6
1.10E+0
6
7.00E+0
5
1.67E+0
7
7.70E+0
6
4.00E+0
6
2.50E+0
6
4.25E+0
7
1.20E+0
7
6.50E+0
6
2.10E+0
6
1.88E+
07
1.02E+
07
2.20E+
06
1.50E+
06
Table 3. Total number of Candida cells calculated per
5ml of original samples at 24, 48, 72, 96 hours of
incubation.
Total # of Candida per 5ml of average 5 initial pure
cultures
8.34E+07
6.39E+07
Tub
e1
2.04
E+0
6
Tub
e2
1.06
E+0
6
Tub
e3
3.34
E+0
6
Tub
e4
8.50
E+0
6
Tub
e5
3.76
E+0
6
1.08
E+0
6
5.40
E+0
5
1.54
E+0
6
2.40
E+0
6
2.04
E+0
6
5.46E+07
4.29E+07
Total # of Candida per 5ml of average 5 initial mixed
cultures
1.87E+07
7.60E+06
3.56E+06
3
1.92E+06
Mean
Variance
Observations
Hypothesized
Mean Difference
df
t Stat
P(T<=t) one-tail
Variable 2 50000000
7945000
40000000
5.71E+13
4
30000000
0
4
5.69205571
0.00235276
20000000
2.13184679
0.00470552
t Critical two-tail
2.77644511
10000000
0
5.00E+07
4.00E+07
3.00E+07
Cells/5ml of sample
6.00E+07
2.00E+07
1.00E+07
0.00E+00
0
24
48
72
Pure Candida
Culture
1
Mixed culture with…
Figure 2. Mean number of Candida cells per 5ml of
samples in Pure and Mixed culture with Lactobacilli.
There is a significant decrease in Candida growth when
mixed with Lactobacilli (P= 0.028767, One tailed
unpaired t-test). Error bars are mean ± SEM.
Using ANOVA and Bonferroni corrections typical
growth of Candida in both pure and mixed cultures
analyzed and results indicated that as incubation hours
increased Growth in all five pure samples and all five
Pure mixed samples followed the same pattern such that
cultur cells growth significantly increased during the first 24
hours of incubation in both cultures. As incubation
e
continued to 48 hours growth dropped significantly and
continued dropping during 72 and 96 hours of
incubation however not as quick as 48 hours. Growth
curve is shown in figure 1.
Mixed P value=0.000112 indicates that growth rate of
cultur Candida is significantly different from 24 through 96
hours of incubations.
e
ANOVA: two-factor without replication
9.00E+07
7.00E+07
60000000
Variable 1
61195000
2.9297E+14
4
t Critical one-tail
P(T<=t) two-tail
8.00E+07
70000000
Cells/5ml of sample
Table 4. Average number of Candida cells calculated
per 5ml of original samples at 24, 48, 72, 96 hours of
incubation.
T-test: two-sample assuming unequal variances
96
Hours of incubation
Figure 1. Average Number of Candida albicans cells
per 5ml of pure culture and mixed culture with
Lactobacilli vs. hours of incubation. There is a
significant statistical Difference in growth of candida
cells throughout the hours of incubation (P=1.12×10 -4,
Anova & Bonferroni correction).
Summary
Row 1
Count
4
Row 2
4
Row 3
4
Column 1
3
Column 2
3
Column3
3
Column 4
3
Sum
240
2.448E+
11
3.178E+
10
1.021E+
11
7.15E+
10
5.816E+
10
4.48E+
10
Average
60
6.12E +
11
7.945E +
09
3.40E +
10
2.38E +
10
1.94E +
10
1.49E +
10
Variance
960
2.9286E
+20
5.7106E
+19
1.91522E
+21
1.21844E
+21
9.33153E
+20
5.87E+
20
4
Discussion
ANOVA:
Source of
variation
SS
df
MS
F
P-value
F Crit
Rows
Columns
8.86E+21 6.01E+20
2
3
4.43E+21 2.00E+20
59.2506 2.680985
1.12E-04 1.40E-01
5.143253 4.757063
It was noted that throughout the incubation, cultures
containing Lactobacilli were more strongly acidic
(pH=5.0-4.0) than pure cultures of the yeasts (pH=6.57.0). This suggested the possibility that the decrease in
C. albicans in mixed culture with Lactobacilli is due to
lactic acid accumulation by Lactobacilli, which inhibits
growth of yeast significantly. To determine the
specificity of the effect, pH determined after 24, 48, 72
and 96 hours of incubation and results are shown in
figure3.
8
7
6
Pure
Candida
Culture
pH
5
4
3
Mixed
culture
with
Lacrobaci
li
2
1
0
0 24 48 72 96
Hours of incubation
Figure 3. pH of mixed MRS broth consist of both yeast
and Lactobacilli and pure thioglycollate broth of
Candida Vs. hours of incubation. Cultures containing
Lactobacilli are more acidic than pure Candida culture
due to lactic acid production by Lactobacilli.
Lactobacillus decreases the growth rate of Candida
albicans yeast by acidifying the environment.
Lactobacillus is a bacterium that produces lactic acid in
its environment so that it will inhibit the growth of
harmful bacteria. Because of lactic acid being the byproduct of lactobacillus it stabilizes the pH of the
vagina, which prevents growth of Candida albicans
(Jeavons, 2003). Candida albicans is a yeast, which
grows in mouth and vagina of humans and infects its
location.
Candida is a fungus that feeds on carbohydrates and
protein such as glucose in order to grow and produce.
When candida starts growing in the vagina, it will
produce mycelia form of hyphae on the lining of the
skin. As it feeds on carbohydrates its will start to grow
and move towards the intestines. When grown, it will
build up a biofilm throughout the body from the vagina
to the intestines. To break down and kill the Candida a
colon-cleansing product will get produced in the
intestines to acidify the environment and deprotonate
Candida albicans (copyright, 2012).
As said before, candida cannot grow in an acidic
environment; its growth factors will be affected.
Moreover, when the bacteria and the yeast are grown in
the same environment, the bacteria (lactobacillus) will
pre-dominate over the yeast (Candida albicans).
In this study we looked at two environments of pure
candida and mixed candida with lactobacilli. The
factors that were measured in the 24, 48, 72, and 96hrs
were the pH, the amount of yeast growth in both
cultures.
As predicted the growth of candida will decrease in the
presence of lactobacillus. As the results show, there was
a decrease in the growth of candida in the mixed
culture because of the presence of lactobacilli. The
more lactobacilli was added to the environment the
more it effected the growth factors of candida. The
reason is because lactobacillus will produce lactic acid,
which will drop the pH of the environment and will be
acidic.
Acknowledgement
We would like to have a special thanks to professor
Teh and the Department of biological science at
Saddleback College for helping and supporting us
throughout our study.
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