ISOLATION, GROWTH, AND MORPHOLOGICAL CHARACTERIZATION OF E.

coli AND BACTERIA

FROM NIMBB WALL
Laurence Christian C. Benig
National Institute of Molecular Biology and Biotechnology
University of the Philippines, Diliman, Quezon City
February 29, 2016
Abstract
The study aims to characterize E.coli based on the effect of different condition in its
growth. Unknown environmental sample was also subjected to the conditions
which will help characterizing the sample. Both E.coli and the environmental
sample were characterized based on their morphology.The E.coli culture and the
environmental sample were diluted first before plating. The plated samples were
then subjected to different conditions; temperature, pH, UV, osmotic pressure,
presence of antiseptics. The samples were also categorized via staining (simple
and Gram).

Keywords: E.coli, Gram-stain, antiseptics, temperature, pH, UV, osmotic pressure

I. Introduction
By pure culture, it is understood as a culture that consists of individuals that came from a single
cell (Avery, 1927). To make the work with bacteria reliable, many efforts were used to devise a reliable
method to isolate a single bacterium.
To help visualizing the cells better, cell staining is a technique that can help. Staining is a
technique that can reveal in vivid detail much information about a cell (Robertson, 1978). A special kind of
staining that is widely used is Gram-staining. Gram-staining differentiates gram-positive and gramnegative bacteria. Gram-positive bacteria retain the iodine-crystal violet complex that is formed during the
staining (Rollins & Joseph, 2000).
Bacterial growth curve is usually defined as the increase in numbers of vegetative cells
(Pommerville, Alcamo, & Alcamo, 2013). Bacterial growth follow a predictable pattern visualized as
population growth curve. Under different conditions, the growth curve will change depending in the effect
of that certain condition.

II. Materials and Methods

A. Serial Dilution

One mililiter of the overnight bacterial suspension was inoculated into a 9 mL saline solution
(0.9% NaCl) in a tube. The solution was mixed by pipetting in and out carefully. Extra measure was taken
to prevent the cotton plug of the pipette to touch the solution. Upon complete mixing, 1 mL of the solution
was transferred into another tube with 9mL saline solution and was mixed. From this second solution, 1
mL was transferred into another tube. The dilution was repeated until the desired dilution was achieved.
Aseptic technique was maintained while diluting.
After the dilutions, the sample was plated. Pour plating was accomplished within a 20-minute
interval between the plating and serial dilutions. Spread plating on the other hand was finished within a
15-minute interval between the plating and serial dilutions.
B. Spread Plating
A sterile pipette tip was used to take 0.1 mL (100 L) of the diluted suspension. It was dropped
evenly onto the corresponding plate. A glass spreader was used to spread the inoculum evenly on the
agar. The plate was then incubated in an inverted position until the next day.
The glass spreader that was used was dipped into an ethanol solution and was flamed. Before
touching the plate, the spreader was cooled by holding it still for about 30 seconds.
C. Pour Plating
In a Petri plate, 1 mL of corresponding dilution was transferred. After making sure it was cool
enough (~45 C), 19 mL of agar was mixed with the dilution in the plate. The plates were incubated upon
hardening in an inverted position at 37 C until the next day.
D. Isolation of Pure Cultures
Three colonies were chosen from the mixed culture plate. Small colony inoculums was
suspended in 1 mL LB broth (CONDA, 10 g/L Bacto-tryptone, 10 g/L NaCl, 5g/L Yeast Extract, 15 g/L
Agar, 750 mL dH2O). Streak plate was done for each group. The plates were incubated in an inverted and
were observed for microbial growth after 24 hours.
E. Simple Bacterial Staining
With a flame-sterilized loop, a colony of bacterial cells (E. coli, B. subtilis, unknown) was touched
on the given plate. The cells were dispersed on a drop of water on a clean slide. The slide was heat-fixed
by passing it briefly over the flame of an alcohol lamp. A drop of methyl blue was added onto the cells.
The slide was then covered with a cover slip.
F. Gram Staining
Bacterial cells were smeared on a slide with water droplets. After spreading the cells, the slide
was heat-fixed. The slide was then flooded with crystal violet. The stain was held for 1 minute before
washing it with water. Iodine was used to stain next for 1 minute before rinsing and draining.
Decolorization was done using 95% ethanol until there was no more crystal violet (5-8 seconds). Lastly,
safranin was used to counter stain for 1 minute before rinsing. Excess water was blot off using tissue.
G. Observation Under the Microscope

The prepared bacterial slides were observed using Nikon SE Microscope under different
objectives. Observations from the microscope were drawn on the lab notebook. For Gram-stained slides,
after focusing on the LPO (Low power objective, a drop of oil was added on top of the slip and then the
objective was shifted to OIO (Oil immersion objective). The same was done to the fungi slide.
H. Monitoring Microbial Growth
Using the given microbial culture, 500 L of bacteria was transferred into a flask containing LB
broth. The flask was incubated at 37 C with shaking.
Serial dilution was done using 100 L aliquot of bacterial suspension. Dilutions 10 -6 and 10-7 was
spread plated using 100 L. With 500 L aliquot of the bacterial suspension, OD 600 was read using
Thermoscientific Nanodrop 2000c UV-Vis spectrophotometer. The reading was done three times and was
averaged. For every 30 minutes, the serial dilution, spread plating, and measuring of OD 600 were done
until the 5th hour.
I. Effect of Temperature
Spread plate of 100 L of the 10 -6 dilution of E. coli suspensions was done into 3 LB agar plates.
The plates were incubated in an inverted position at 4 C (plate 1), room temperature (~25 C, plate 2),
and at 37 C (control plate). A 4th plate was incubated in an inverted position at 4 C. The sample in the 4 th
plate was treated with moist heat using an autoclave prior to plating.
J.

Effect of pH
From the overnight bacterial culture, 500 L was transferred into a flask containing 50 mL of LB

agar pH 9. The plate was incubated in an inverted position at 37 C with shaking. Every 30 minutes, OD 600
was measured using the same procedure as in I.
K. Effect of Osmotic Pressure
From the overnight bacterial culture, 500 L was transferred into a flask containing 50 mL of LB
agar 5% and 10% NaCl. Every 30 minutes, OD600 was measured using the same procedure as in I.
L. Effect of UV Irradiation
From the E.coli suspension, 100 L of 10-6 was spread plated into 3 LB agar plates. The plates
(1-3) were exposed to UV light for 5, 10, and 15 minutes respectively. The plates were incubated in an
inverted position at 37 C overnight. The number of colonies were counted and the resulting growth ere
compared the next day.
M. Effect of Antiseptics
An LB agar plate with 200 L of E. coli suspension was lawn-inoculated. Filter discs were then
soaked in antimicrobial agents of known concentration. The soaked filter discs were placed on the plates;
equal spacing between each was made sure. The diameters of the inhibition zone were measured.

III. Results
Table 1 Number of Colonies per plate
Dilution

Number of Colonies

Spread
10-1
10-2
10-3
10-4
10-5
10-6
10-7
Blank 10-1
Blank 10-2

Pour

Trial 1
TMTC
TMTC
TMTC
TMTC
276
91
7
None
1

Trial 2
TMTC
TMTC
TMTC
TMTC
276
41
11
None
4

Trial 1
TMTC
TMTC
TMTC
TMTC
TMTC
251
32
52
None

Trial2
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
48
6
None

Table 2 Number of Colony-forming units per mL (CFU/mL) per plate

Calculated CFU/mL
Dilution
-1

10
10-2
10-3
10-4
10-5
10-6
10-7
Blank 10-1
Blank 10-2
Table 3

Spread
Trial 1
TMTC
TMTC
TMTC
TMTC
2.76x108
9.10x108
7.00x108
N/A
1.00x103

Pour
Trial 2
TMTC
TMTC
TMTC
TMTC
2.76x108
4.10x108
1.10 x109
N/A
4.00x103

Trial 1
TMTC
TMTC
TMTC
TMTC
TMTC
2.51x109
3.20x109
5.20x103
N/A

Trial2
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
4.80x109
6.00x102
N/A

Arithmetic and Weighted Average of Calculated Colony-forming units per mL (CFU/mL)

per plate
Calculated CFU/mL
Spread Plate
4.68x108
3.39x106

Average Mean
Weighted Mean

Pour Plate
3.50x109
2.76x108

Table 4 Colonial morphology of different specimens.

Specimen
Shape

Edge

Color

Surface

Elevation

Gram

B. subtilis

circular

entire

creamy white

smooth

raised - flat

Positive

E. coli

circular

undulate

clear

smooth

raised

Negative

S. aureus

circular

entire

creamy white

smooth

raised- convex

Positive

S. cerevisiae circular

entire

faint yellow

smooth

convex

N/A

P20 Bill samplecircular

entire

orange

smooth

raised

Negative

MBB wall samplecircular

entire

white

smooth

flat

Positive

entire

white

smooth

raised

Positive

Unknown

circular

Table 5 OIO View of Different Environmental Samples Stained with Methyl Blue and Gram Stain
Gram stain

Methyl Blue

Environmental: P20 Bill

Environmental: NIMBB Wall

Unknown

Table 6 Measured OD600 and Calculated Cell/mL for pH 9 and pH 3

pH 9

pH 3

OD600

Cells/mL

OD600

Cells/mL

1.60 x10-2

1.28 x107

2.13 x10-2

1.71 x107

0.5
1
1.5
2
2.5
3
3.5
4
4.5

2.5 x10-2
3.13 x10-2
1.9 x10-2
0.12 x10-2
-1.7 x10-2
-2.67 x10-2
2.67 x10-2
-2.80 x10-2
2.40 x10-2

2.00 x107
2.51 x107
1.52 x107
0.96 x107
-1.40 x107
-2.10 x107
2.13 x107
-2.20 x107
-1.9 x107

2.63
2.86
3.30
0.67
1.27
1.10
17.8
3.23
0.13

x10-2
x10-2
x10-2
x10-2
x10-2
x10-2
x10-2
x10-2
x10-2

2.11
2.29
2.64
0.53
1.01
0.88
14.2
2.59
0.11

x107
x107
x107
x107
x107
x107
x107
x107
x107

Table 7 Measured OD600 and Calculated Cell/mL for pH 9 and pH 3

10% NaCl

0
0.5
1
1.5
2
2.5
3
3.5
4
4.5

OD600
2.40 x10-2
2.83 x10-2
3.90 x10-2
5.17 x10-2
1.9 x10-2
0.10 x10-2
0.2 x10-2
0.43 x10-2
0.93 x10-2
1.23 x10-2

5% NaCl

Cells/mL
1.92 x107
2.27 x107
3.12 x107
4.13 x107
1.52 x107
0.08 x107
0.16 x107
0.3 x107
0.75 x107
0.99 x107

OD600
2.33 x10-2
2.60 x10-2
2.23 x10-2
4.83 x10-2
1.40 x10-2
8.13 x10-2
0.85 x10-2
6.83 x10-2
10.7 x10-2
12.3 x10-2

Cells/mL
1.87 x107
2.08 x107
1.79 x107
3.87 x107
1.12 x107
6.51 x107
0.68 x107
5.47 x107
8.56 x107
9.87 x107

Cells/mL vs Time
600000000
500000000

10% NaCl

400000000
Cell's/mL

5% NaCl

300000000

pH 9

200000000

pH 3

100000000

LB Broth

0
-100000000 0

Time (Hours)

Figure 1 Cells/mL (in millions) vs Time Graph

Table 7 Effects of UV and Temperature in Microbial Growth

Temperature

UV

(4 C) No visible colony

(20 minutes) 32

(25 C) No visible colony

(40 minutes) 33

(37 C) 80

(60 minutes) 12

Table 8 Zone of Inhibition of Different Antibiotic/Natural Extract

Amoxicillin
Terramycin
Negative
Terramycin

Plate 1

Plate 2

No visible zone of inhibition

No visible zone of inhibition
No visible zone of inhibition

No visible zone of inhibition

No visible zone of inhibition
No visible zone of inhibition

IV. Discussion
Isolation of Pure Culture
Theoretically, the culture isolated from the plates should be pure. The sample is grown in a
medium in such a way that the individual cells across the medium surface. Since the individual cells are
separated they will create a discrete colony when they multiply which may then be used to inoculate more
medium with the assurance of only one type of organism is present.
Before plating, bacterial culture should first be diluted. Since there millions of organism in a
culture, counting manually is impossible. Serial dilution is commonly done to help in reducing the number
of organism in a plate (MacLowry, Jaqua, & Selepak, 1970). After counting the number of colony, one can
estimate the number of organism in the original sample using the dilution factor.
It is a necessity for the agar in pour plating to be cooled to about 45 C. Not doing so might result
in lower count due to the heat-sensitive bacteria dying (Hoben & Somasegaran, 1982). The spreader
used in spread plating needs to be cooled before touching the agar or the culture. Using the spreader
while it is still hot will result in destroying the agar and in the cultures case, will result in the bacteria
dying.

Once the culture is isolated, the purity of the isolate is needed to be determined to know whether
the isolation is successful. Low purity suggests that the isolation method is not effective. But if the
method has shown success in the past, contamination should be considered as a cause for low purity.
Contamination might occur if the method is not done aseptically.
A study done by Hoben and Somasegaran suggests that pour plating and spread plating can be
interchangeable. Based on the data in table 1, we can say that spread plating is more precise than pour
plating in isolating bacterial culture. In another study done by Taylor, Allen, and Geldreich in 1983
concludes that pour plate is neither as accurate nor as precise as spread plating which agrees with the
data. However, they also conclude that pour plating can still be used as an alternative to spread plating.
Microbial Morphology and Bacterial Staining
Gram-stained sample data agrees with the expected results. Bacillus subtilis and Staphylococcus
aureus is gram positive while Escherichia coli is gram negative. Gram staining differentiates bacteria
based on their ability to retain the crystal violet dye during solvent treatment. In gram positive bacteria,
their cell walls block the iodine-crystal violet complex (Rollins & Joseph, 2000)
Effects of Different Conditions in Bacterial Growth
Based on Figure 1 and Table 7, increasing the concentration of salt will increase the osmotic
pressure which will result in decrease in growth of the bacteria. In 1991, Houssin, Eynard, Shechter, and
Ghazi conducted a study in which the effect of osmotic pressure on E. coli is the concern. They found out
that osmotic upshock resulted in large decrease in growth of the bacteria.
The plates were placed in three different temperatures to study effect of different temperature in
bacterial growth. In a study done by the group of Pothakamury, Vega, Zhang, Barbosa-Canovas, and
Swanson, they found out that increasing the temperature resulted in an exponential decrease in bacterial
growth.
Antibiotic-soaked filter discs were placed with enough distance between to make sure that the
inhibition zone that will occur is cause by one antibiotic. In a study made by Baker and Pulaski, they found
out that treating fecal samples with terramycin will eliminate E. coli. In the experiment, the terramycin
treatment did not produce any inhibition zone. The effect of antibiotic in bacteria points toward a
interference with protein metabolism of the organism (Hahn & Wissenman Jr., 1951).
The growth of E.coli decreased 10-to-100 fold as the pH was increased to pH 8 (Small,
Blankenhorn, Welty, Zinser, Slonczewski, 1994). Agreeing with the previous study, the bacterial growth in
the medium with pH 9 is far less than the regular medium (Figure 1).

In the experiment prolonged exposure in UV inhibits the growth of E.coli. Witkin, in his study in
1976, said that UV owes its mutagenic effect in E. coli to misrepair of damaged chromosome.

V. Summary & Conclusion

The study aimed to characterize E. coli and an environmental sample in terms of effect of
different conditions and staining. E. coli was stained and was found to be gram-negative which agrees
previous studies. The growth curve E. coli normally lasts for 8 hours. The different conditions the E. coli
was subjected to reduce that time. Increasing the osmotic pressure by increasing NaCl concentration led
to decrease in growth of the bacteria. The same result was observed while studying the effects of
temperature and pH. Increasing the pH to more than 8 led to an exponential decrease in growth (Small, et
al, 1994). Prolonged exposure to UV also decreases the growth of the bacteria. Presence of antiseptics
inhibits the growth of the bacteria in an area surrounding the antiseptic.
The environmental sample (NIMBB wall) was found out to be a circular, with entire edge, white,
smooth, Gram-positive bacteria. The other environmental sample (P20 bill) was found out to be a circular,
with entire edge, white, smooth, raised, Gram-negative bacteria.

VI. References
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