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Michael J. Wallach II
11/6/2009
Dr. Jack Kennell
General Microbiology Laboratory 465
Fall 2009
Section 37
INTRODUCTION
Bacteria and their ability to cause diseases in a wide variety of forms is one of many reasons for them being
such a research interest. Another reason is their beneficial side as well. Bacteria live in the intestinal track of
humans and all kinds of other animals, digesting molecules the host would have been incapable of absorbing the
nutrients. Either way one looks at it, it is crucial to understand the features that lend to bacterial virility and to
investigate the ways in which bacterial growth can be controlled.
In this unit, Acinetobacter calcoaceticus and Escherichia coli were used to demonstrate the ways in which
virulent genes may be transferred between bacterial species or strains of the same species. Transformation is the
absorption of another bacterium’s DNA or fragment by a recipient bacterium. The DNA molecule is naked and
often very fragile and must be received to the chromosome in an inheritable form.(). A Detergent mediated
transformation required use of sodium dodecyl sulfate (SDS) in saline-sodium citrate (SSC) to prepare a crude DNA
extract. The receiver cells are heated to be and incubated with the DNA for absorption. Enterococcus faecalis,
Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Salmonella typhimurium were used
throughout the lab to also show the effects of Ultraviolet non-ionizing radiation on growth, antibiotics’ effects via
the Kirby-Bauer Technique, heat’s effect on growth, and finally how common disinfectants from everyday life, such
as bleach and Clearasil, restrict growth. Disinfectants are chemical agents used on inanimate objects to lower the
amount of microbes on the surface.()
Broad-spectrum, ones that affect a wide-range of organisms, and narrow-spectrum, ones that affect a select
group of organisms, were tested.().
All inoculation on both solid and liquid media utilized aseptic technique, sterilizing the inoculating loop
with a Bunsen burner flame both before and after contact with a specimen or media surface. When using
micropipettes, a new pipette tip was used for contact with every specimen, broth, or surface. For inoculating solid
media, the sterilized loop was either dipped into a liquid culture or used to obtain a visible sample from a solid agar
surface. The loop was then streaked across the intended area of the surface, with the loop parallel to the surface.
All agar plates to be incubated, and sections if applicable, were labeled with the group members’ initials, date, lab
section, media type, and inoculant. All eppendorf tubes were labeled with the initials of the contained material or
specimen. All agar plates were inverted for incubation.
Bacterial Transformation
Using aseptic technique, a visible sample of streptomycin-resistant Acinetobacter calcoaceticus (Strr)
was transferred using an inoculating loop from a prepared BHI agar plate culture to an eppendorf tube containing
500µL of sodium dodecyl sulfate (SDS) in saline-sodium citrate (SSC). The cells were suspended in the SDS in
SSC liquid by pipetting up and down carefully with a micropipette. The eppendorf tube was then placed in a tube
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rack in a prepared 60ºC water bath and incubated for 30 minutes. A smaller amount of Strr and streptomycin-
sensitive (Strs) A. calcoaceticus was aseptically transferred with an inoculating loop from respective prepared BHI
agar plate cultures to separate sections 1 and 2 of a labeled BHI agar X-plate. A second, visible loop-full of Strs A.
calcoaceticus from the BHI agar culture was aseptically added to section 3 of the BHI agar X-plate with an
inoculating loop. Each sample was streaked in the center of the respective section in a circle of approximately 1cm2.
Following incubation of the eppendorf tube, an inoculating loop was used to aseptically suspend the Strr A.
calcoaceticus DNA in the tube and mix a loop-full with the 1cm2 streak of Strs cells in section 3 of the BHI agar X-
plate. A control loop-full of the DNA was aseptically streaked on section 4 of the BHI agar X-plate in the same
1cm2 sized manner. The BHI agar X-plate was incubated at 30ºC for approximately 24 hours. Observations were
recorded. Following incubation, sections 1-3 of a new BHI agar X-plate containing streptomycin sulfate was
labeled identically to sections 1-3 of the incubated BHI agar X-plate. Using an inoculating loop, a small amount of
culture from the center of sections 1-3 of the incubated BHI agar X-plate was aseptically streaked onto their
respective sections on the BHI agar X-plate containing streptomycin in a 1cm2 circle. Section 4 of the new BHI agar
was left blank. The old BHI agar X-plate was refrigerated and the new BHI agar X-plate with streptomycin was
incubated at 30ºC for approximately 24 hours. Observations were recorded.
Prepared competent Escherichia coli cells were taken from a -80ºC freezer and allowed to thaw in an open-
to-room-temperature ice-filled cooler. Using a micropipette, 20µL of the cells were transferred and suspended in a
1.5mL eppendorf tube with 0.1µg of a prepared plasmid DNA mixture. The mixture contained both Lac Z+ and Lac
Z- plasmids. The tube was incubated on ice for 20 minutes. Following incubation, the tube was placed in a 42ºC
water bath for 45 seconds. 500 µL of Lysogeny broth (LB) was then added with a micropipette and mixed in the
same manner as suspension. The tube was incubated at 37ºC for approximately 20 minutes. After incubation,
200µL of the cell suspension in the eppendorf tube was spread onto an LB agar plate containing ampicillin (amp)
and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal). To spread the cell suspension, the 200µL was first
transferred aseptically from the tube to the center of the LB/ amp/ X-gal plate with a micropipette. A new, sterile
plate spreader was then held in place while the plate spinner was spinning the agar to spread the suspension evenly
over the surface. Once dry, the plate was incubated at 37ºC for approximately 24 hours. Results were observed and
colonies were counted.
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Bacillus subtilis, E. coli, and Salmonella typhimurium in place of P. aeruginosa. This resulted in a total of 24 trials.
After incubation, all plates were observed and zones of inhibition around the disks were measured in millimeters.
To measure the zone of inhibition, a small ruler was used to measure, to the nearest millimeter, the total diameter of
the antibiotic coated disk and area of no growth, if present. All measurements were recorded and the plates were
incubated for another 24 hours at 37ºC.
RESULTS
Bacterial Transformation
Bacterial growth was observed in all four sections of the BHI agar X-plate after 24 hours of incubation.
The Str A. calcoaceticus streaked in section 2 grew along the inoculated 1cm2 circle. The Strs A. calcoaceticus
r
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streaked in section 1 produced a lawn spanning the entire section with a darker, cream colored center along the 1cm2
circle of inoculant. The exterior portion of the lawn appeared filamentous and partially translucent. The Strs A.
calcoaceticus streaked with the crude Strr A. calcoaceticus DNA on section 3 yielded similar growth to that of
section 1. It too had a lawn of growth of similar color and shape spanning the entire section with a darker cream
colored center along the 1cm2 circle of inoculant. Section 4 containing the Strr DNA control streak produced very
little to no growth along what appears to be heavy inoculant. The second BHI agar X-plate containing the
streptomycin sulfate showed growth on 2 of the 3 inoculated sections following the 24 hours of incubation . Strs A.
calcoaceticus lacked any growth on section 1. This is clear as only the thick 1cm2 inoculant can be seen. The Strr
A. calcoaceticus streaked onto section 2 yielded a thick growth along the inoculated area. It is a dark cream colored
cluster of colonies with a darker cream color in denser areas. Section 3 streaked with Strr A. calcoaceticus DNA and
Strs A. calcoaceticus cells yielded a significant amount of cream colored growth. This is along the area of streaking
and seems to show many colonies almost isolated in the interior. This growth is not as thick as that seen in section
2, but appears to be of similar color.
Various isolated E. coli colonies were observed to have grown on the LB/ amp/ X-gal agar. A total of 72
total colonies were counted, 68 of which were blue and 4 of which were white. This indicated 94% blue colony
growth. A zoomed in image clearly showing a white colony can be seen in. The colonies all appeared entirely
round in shape with a raised elevation. The blue colonies seemed white to light blue along the circumference and
dark blue in the center. The white colonies had continuous color.
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has two lower measurements of 13.5mm from bacitracin and 12mm from nalidixic acid. The higher measurement
range is from 15.5mm from streptomycin to 38mm from amoxicillin. B. subtilis had the largest overall zones of
inhibition on MHII and BHI, with only 6mm from bacitracin being the lowest and the highs ranging from 23mm
from sulfisoxazole to 38.5mm from cefazolin on MHII. On BHI agar, the bacitracin-caused zone of inhibition was
recorded as 2.5mm with the others ranging from 18.5mm from nalidixic acid to 39mm from amoxicillin. E. faecalis
was observed to have the most variable zones of inhibition. On MHII agar sulfisoxazole, streptomycin,
sulfamethoxazole/ trimethoprim, and nalidixic acid caused the small zones of 6mm, 10mm, 12.5mm, and 13mm
respectively. The larges zones were ranged 15mm from bacitracin to 25.5mm tetracycline. On BHI agar, E.
faecalis had 3 small 8mm zones inhibition from sulfisoxazole, nalidixic acid, and streptomycin. The rest of the
antibiotics produced large zones ranging from 18mm from cefazolin to 32.5mm from sulfamethoxazole/
trimethoprim.
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growth at 30 seconds in both B. cereus and E. coli, and at 90 seconds in S. aureus. Listerine with 0.64% thymol
eliminated growth in 30 seconds in B. cereus and E. coli but not until after 90 seconds in S. aureus, with moderate
growth (2) still at 90 seconds. 3% H2O2 restricted B. cereus growth to moderate (2) at 30 seconds and completely
restricted it at 90 seconds. 3% H2O2 caused an almost linear rate of growth restriction in S. aureus and E. coli, both
still at moderate (2) growth at 5 minutes of exposure. The First aid wash with 0.13% Benzalkonium chloride
completely restricted growth in S. aureus and E. coli at 30 seconds, and in B. cereus at 90 seconds. The 1:100
dilution hand soap did not restrict growth in B. cereus and E. coli, and barely restricted growth of S. aureus to a 3 at
90 seconds exposure. The 1:100 dilution of Clearasil with 0.3% triclosan did not restrict growth of E. coli, but at 30
seconds completely restricted (0) the growth of B. cereus and severely restricted(1) the growth of S. aureus. At 90
seconds, S. aureus growth was completely restricted (0). A 1:5 dilution of Neosporin showed some immediate
growth restriction (3) at 30 seconds in S. aureus and E. coli, with growth further being restricted at 5 minutes of
exposure. B. cereus initially showed some susceptibility to the Neosporin dilution reducing to a growth number of
2, but by 5 minutes of exposure growth was still no further restricted. Contaminations or experimental accidents are
noted with a * in Table2.
DISCUSSION
Bacterial Transformation
When performing the SDS in SSC mediated transformation, the growth obtained in the Strs A.
calcoaceticus inoculated section 1 of the first BHI agar X-plate had a filamentous lawn of bacterial cells over the
entire surface . At the center was a denser, cream colored circular colony cluster surrounded the approximately
1cm2 inoculating streaks. When compared to the Strr A. calcoaceticus inoculated section 2, it was observed that the
circular center on the Strs section shared more morphological features with the Strr section than the filamentous
lawn. This indicates a contamination present. In section 4, the Strr DNA control streak showed signs of slight
growth indicating another possible contaminant or possible incomplete lyses in the water bath. Both contaminants
were not presumed to be the same as severe differences in growth amount occurred between the two on the BHI
agar. Section 3 inoculated with Strr A. calcoaceticus DNA and Strs A. calcoaceticus seemed to yield the same
filamentous contaminant as section 1. This indicates a possible contaminant on the prepared Strs A. calcoaceticus
BHI agar sample. Final results obtained were typical and as expected. The second BHI agar X-plate contained
streptomycin sulfate. Strs A. calcoaceticus lacked growth in section 1 because it lacked a streptomycin-resistance
gene, much like that in the Strr A. calcoaceticus genome. (). This was why Strr A. calcoaceticus grew in section 2.
The growth in section 3 of the Strr A. calcoaceticus DNA - Strs A. calcoaceticus inoculant was because the Strs strain
had been successfully transformed into carrying the Strr strain gene fore streptomycin resistance.().
The Heat-Shock results obtained were as expected . No contaminant was observed and blue and white
colonies were well isolated. Blue colonies were the result of 94% of the colonies. Blue colonies indicate that the E.
coli has the pBlu plasmid DNA because Isopropyl-β-D-thio-galactoside (IPTG) induced the lacZ gene to produce a
functional ß-galactosidase. This functional protein cleaves the X-gal in the media causing the colony to turn blue.().
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In the white colonies, the E. coli has the pAmp plasmid DNA. Hence, no functional ß-galactosidase is induced by
IPTG and X-gal cannot be cleaved. No blue metabolites are produced.
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synthase and thus the block of dihydrofolate reductase by trimethoprim is needed. Chloramphenicol inhibits
proteins synthesis by blocking the RNA’s ability to assemble the amino acids. It is a wide-spectrum antibiotic and
was shown effective against all tested organisms except P. aeruginosa. (). P. aeruginosa most likely has a drug
efflux system for chloramphenicol. Amoxicillin was effective against S. aureus, E. coli, and S. typhimurium. More
experiments on a highly effective antibiotic for P. aeruginosa would prove beneficial.
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REFERENCES
Kennell, Jack. "Nutrition, Culturing, and Growth." Microbiology 464-01. Saint Louis: Saint Louis University, 2009.
Köhler, T, et al. "Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas
aeruginosa." PubMed 40 (1996): 2288-2290.
Leboffe, Michael J and Burton E Pierce. A Photographic Atlas for the Microbiology Laboratory. Ed. David
Ferguson. 3rd. Englewood: Douglas N. Morton, 2005.
Roe, Bruce A. Protocol Book. 4 December 2007. 05 November 2009
<http://www.genome.ou.edu/protocol_book/protocol_adxF.html>.
Simmons, Christine. "An Introduction to Antimicrobials." Simmons, Christine. General Microbiology Laberatory
Manual. Saint Louis: Saint Louis University Department of Biology, 2009. 8.
—. General Microbiology Laboratory Manual. Saint Louis, 2009.
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