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 gram-

negative 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 µL) 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 ºC), 19 mL of agar was mixed with the dilution in the plate. The plates were incubated upon

hardening in an inverted position at 37 ºC 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 µL of bacteria was transferred into a flask containing LB

broth. The flask was incubated at 37 ºC with shaking.

Serial dilution was done using 100 µL aliquot of bacterial suspension. Dilutions 10 -6 and 10-7 was

spread plated using 100 µL. With 500 µL 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 µL 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 ºC (plate 1), room temperature (~25 ºC, plate 2),

and at 37 ºC (control plate). A 4 th plate was incubated in an inverted position at 4 ºC. 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 µL was transferred into a flask containing 50 mL of LB

agar pH 9. The plate was incubated in an inverted position at 37 ºC 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 µL 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 µL 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 ºC 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 µL 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

DilutionNumber of Colonies

Spread PourTrial 1 Trial 2 Trial 1 Trial2

10-1 TMTC TMTC TMTC TMTC10-2 TMTC TMTC TMTC TMTC10-3 TMTC TMTC TMTC TMTC10-4 TMTC TMTC TMTC TMTC10-5 276 276 TMTC TMTC10-6 91 41 251 TMTC10-7 7 11 32 48

Blank 10-1 None None 52 6Blank 10-2 1 4 None None

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

DilutionCalculated CFU/mL

Spread PourTrial 1 Trial 2 Trial 1 Trial2

10-1 TMTC TMTC TMTC TMTC10-2 TMTC TMTC TMTC TMTC10-3 TMTC TMTC TMTC TMTC10-4 TMTC TMTC TMTC TMTC10-5 2.76x108 2.76x108 TMTC TMTC10-6 9.10x108 4.10x108 2.51x109 TMTC10-7 7.00x108 1.10 x109 3.20x109 4.80x109

Blank 10-1 N/A N/A 5.20x103 6.00x102

Blank 10-2 1.00x103 4.00x103 N/A N/A

Table 3 Arithmetic and Weighted Average of Calculated Colony-forming units per mL (CFU/mL) per plate

Calculated CFU/mLSpread Plate Pour Plate

Average Mean 4.68x108 3.50x109

Weighted Mean 3.39x106 2.76x108

Table 4 Colonial morphology of different specimens.

SpecimenShape 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 sample circular entire orange smooth raised Negative

MBB wall sample circular entire white smooth flat Positive

Unknown circular entire white smooth raised Positive

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

0 1.60 x10-2 1.28 x107 2.13 x10-2 1.71 x107

0.5 2.5 x10-2 2.00 x107 2.63 x10-2 2.11 x107

1 3.13 x10-2 2.51 x107 2.86 x10-2 2.29 x107

1.5 1.9 x10-2 1.52 x107 3.30 x10-2 2.64 x107

2 0.12 x10-2 0.96 x107 0.67 x10-2 0.53 x107

2.5 -1.7 x10-2 -1.40 x107 1.27 x10-2 1.01 x107

3 -2.67 x10-2 -2.10 x107 1.10 x10-2 0.88 x107

3.5 2.67 x10-2 2.13 x107 17.8 x10-2 14.2 x107

4 -2.80 x10-2 -2.20 x107 3.23 x10-2 2.59 x107

4.5 2.40 x10-2 -1.9 x107 0.13 x10-2 0.11 x107

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

10% NaCl 5% NaCl

OD600 Cells/mL OD600 Cells/mL0 2.40 x10-2 1.92 x107 2.33 x10-2 1.87 x107

0.5 2.83 x10-2 2.27 x107 2.60 x10-2 2.08 x107

1 3.90 x10-2 3.12 x107 2.23 x10-2 1.79 x107

1.5 5.17 x10-2 4.13 x107 4.83 x10-2 3.87 x107

2 1.9 x10-2 1.52 x107 1.40 x10-2 1.12 x107

2.5 0.10 x10-2 0.08 x107 8.13 x10-2 6.51 x107

3 0.2 x10-2 0.16 x107 0.85 x10-2 0.68 x107

3.5 0.43 x10-2 0.3 x107 6.83 x10-2 5.47 x107

4 0.93 x10-2 0.75 x107 10.7 x10-2 8.56 x107

4.5 1.23 x10-2 0.99 x107 12.3 x10-2 9.87 x107

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5-100000000

0

100000000

200000000

300000000

400000000

500000000

600000000

Cells/mL vs Time

10% NaCl5% NaClpH 9pH 3LB Broth

Time (Hours)

Cell'

s/m

L

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

Table 7 Effects of UV and Temperature in Microbial Growth

Temperature UV

(4 ºC) No visible colony (20 minutes) 32

(25 ºC) No visible colony (40 minutes) 33

(37 ºC) 80 (60 minutes) 12

Table 8 Zone of Inhibition of Different Antibiotic/Natural Extract

Plate 1 Plate 2

Amoxicillin

Terramycin No visible zone of inhibition No visible zone of inhibition

Negative No visible zone of inhibition No visible zone of inhibition

Terramycin 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 ºC. 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 culture’s 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

Avery, R. C. (1927). A Simple Method For The Isolation Of Pure Cultures From Single Bacterial Cells. Journal of Experimental Medicine, 45(6), 1003-1007.

Baker, H. J., & Pulaski, E. J. (1950). Effects Of Terramycin On Fecal Flora. Annals of the New York Academy of Sciences, 53(2), 324-331.

Hahn, F. E., & Wisseman, C. L. (1951). Inhibition of Adaptive Enzyme Formation by Antimicrobial Agents. Experimental Biology and Medicine, 76(3), 533-535.

Hoben, H. J., & Somasegaran, P. (1982). Comparison of the Pour, Spread, and Drop Plate Methods for Enumeration of Rhizobium spp. in Inoculants Made from Presterilized Peatt. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 44(5), 1246-1247. Retrieved from http://aem.asm.org

Houssin, C., Eynard, N., Shechter, E., & Ghazi, A. (1991). Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli. Biochimica Et Biophysica Acta (BBA) - Bioenergetics, 1056(1), 76-84.

MacLowry, J. D., Jaqua, M. J., & Selapak, S. T. (1970). Detailed Methodology and Implementation of a Semiautomated Serial Dilution Microtechnique for Antimicrobial Susceptibility Testing. Applied Microbiology, 20(1), 46-53. Retrieved from http://aem.asm.org

Pommerville, J. C., Alcamo, I. E., & Alcamo, I. E. (2013). Alcamo's fundamentals of microbiology. Sudbury, MA: Jones & Bartlett Learning.

Robertson, R. T. (1978). Neuroanatomical research techniques.

Small, P., Blakenhorn, D., Welty, D., Zinser, E., & Slonczewski, J. (1994). Acid and base resistance in Escherichia coli and Shigella flexneri: Role of rpoS and growth pH. Journal of Bacteriology, 176(6), 1729-1737. Retrieved from http://jb.asm.org/content/176/6/1729.short

Taylor, R. H., Allen, M. J., & Geldreich, E. E. (1983). Standard plate count: A comparison of pour plate and spread plate methods. American Water Works Association, 75(1), 35-37. Retrieved from http://www.jstor.org/stable/41272873

Zhang, Q., Monsalve-González, A., Barbosa-Cánovas, G. V., & Swanson, B. G. (1994). Inactivation of E. Coli and S. Cerevisiae by Pulsed Electric Fields Under Controlled Temperature Conditions. Transactions of the ASAE, 37(2), 581-587.