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).
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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).
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.