Identification of Streptomycin-Resistance Transformation Followed by Observation of Various Antibiotic, Antiseptic, and Disinfectant Susceptibilities on Selected Bacteria

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. (Simmons, General Microbiology

Laboratory Manual 73). 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. (Simmons, General

Microbiology Laboratory Manual 85)

Broad-spectrum, ones that affect a wide-range of organisms, and narrow-spectrum, ones that affect a select

group of organisms, were tested. (Simmons, General Microbiology Laboratory Manual 79).

MATERIALS & METHODS

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.

Antibiotic Susceptibility Testing

A new, sterile cotton swab was dipped into a Pseudomonas aeruginosa broth and streaked thickly onto a

labeled Mueller–Hinton (MHII) agar plate using a 4-streak method. This was repeated for an additional labeled

MHII agar plate. Disk dispenser 1 was used to stamp 5ug kanamycin (K), 25ug sulfisoxazole (G), 30ug

chloramphenicol (C), 30ug amoxicillin (AmC), 30ug nalidixic acid (NA), and s10ug treptomycin (S) coated disks

onto the first MHII agar plate with the lid off. Dispenser 2 was used to stamp 30ug tetracycline (Te), 10units

penicillin (P), 15 ug erythromycin (E), 23.75ug and 1.25ug of sulfamethoxazole/ trimethoprim (SxT), 30ug cefazolin

(CZ), and 10 IU of bacitracin (B) coated disks onto the second MHII agar plate with the lid off. This entire

procedure was repeated for a second trial. The plates were incubated at 37ºC for 24 hours. This was once again

repeated but for BHI agar plates. All prior steps were repeated for Enterococcus faecalis, Staphylococcus aureus,

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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.

Physical and Chemical Methods of Control

A prepared culture of S. aureus and a culture of Bacillus cereus were used to aseptically transfer and streak

each organism with an inoculating loop onto its own section 1 of 2 nutrient agar X-plates. Both prepared cultures

where then incubated for 10 minutes in a 40ºC water bath. Following incubation, both organisms were once again

inoculated onto their respective nutrient agar X-plates with an inoculating loop on section 2. This process was

repeated for incubation in an 80ºC water bath and streaked onto section 3. Both agar plates were incubated at 37ºC

for 48 hours.

Prepared cultures of B. cereus, E. coli, and S. aureus were used to aseptically transfer a loop-full of

inoculant from the culture broths and streak them onto separate sections on 1 nutrient agar Y-plate. This was

repeated for 2 more nutrient agar Y-plates. One plate was labeled as the control and incubated at 37ºC for 48 hours.

The second nutrient agar Y-plate had its lid removed, was inverted, and then placed on a UV light box covered by a

clear door for 2 minutes. This was repeated for the third nutrient agar Y-plate for 5 minutes. Both plates were also

covered and incubated, along with the control, for 48 hours at 37ºC.

One section of a nutrient agar X-plates was divided in half with a permanent marker for the two controls,

creating five total sections. 70% ethanol was inoculated onto section A using aseptic technique and labeled as the

disinfectant control. A loop-full of B. cereus liquid culture was inoculated onto section B using aseptic technique

and labeled as the organism control. With a micropipette, 500µL of B. cereus liquid culture was added to the 70%

ethanol eppendorf tube and mixed gently by tapping the bottom and side. A stop-clock was set and after 30 seconds,

a loop-full of the disinfectant/ organism mixture was inoculated onto section C. After 90 seconds, a loop-full of the

disinfectant/ organism mixture was inoculated onto section D. After 5 minutes, a loop-full of the disinfectant/

organism mixture was inoculated onto section E. All sections were additionally labeled with their corresponding

time periods. This process was repeated for S. aureus and E. coli. Sterile water, 1% phenol, Betadine, 10% bleach,

1% bleach, Listerine, 3% H2O2, antiseptic lidocaine hydrochloride, 62% ethyl alcohol hand sanitizer, foaming

antiseptic wash, 1:100 diluted hand soap, 1:100 diluted Clearasil, 70% isopropyl alcohol, and 1:5 diluted Neosporin

were all also used instead of 70% ethanol for all three organisms. This resulted in a total of 45 inoculated nutrient

agars. All plates were incubated at 37ºC for 48 hours. Observations were recorded.

RESULTS

Bacterial Transformation

Bacterial growth was observed in all four sections of the BHI agar X-plate after 24 hours of incubation.

The Strr A. calcoaceticus streaked in section 2 grew along the inoculated 1cm2 circle. The Strs A. calcoaceticus

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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.

Antibiotic Susceptibility Testing

Detailed data for every trial is listed in Table 1. The trials are averaged and graphed by organism in Chart

1 for MHII agar and Chart 2 for BHI agar. Average zones of inhibition are labeled. The smallest possible

measurement indicating no zone of inhibition is 6mm, the width of the disk. This zone of inhibition caused by the

tested antibiotic compounds on the implanted disks seemed to hardly affect P. aeruginosa overall, especially when

compared to the other organisms tested. This held true on both BHI and MHII agar. Largest zones of inhibition

where seen for tetracycline and streptomycin with 13mm and 12.5mm receptively on MHII agar and 15mm and

13mm on BHI agar. On MHII agar, growth around the erythromycin disk was partially halted, thinning out

progressively towards the disk. This thinning region begins a large distance away from the disk. For S.

typhimurium, larger zones of inhibition where witnessed ranging from 15.5mm from streptomycin, to 28mm from

cefazolin on MHII agar . On BHI agar these zones were increased with almost all but bacitracin, erythromycin, and

penicillin in the range of 16.5mm from streptomycin to 32mm from sulfamethoxazole/ trimethoprim. These three

outside the range measured 7mm, 12.5mm, and 12mm respectively. Similarly for E. coli, only bacitracin,

erythromycin, and penicillin measuring 7mm, 9.5mm, 7mm respectively were outside a range of 18mm from

amoxicillin to 25mm from cefazolin, sulfamethoxazole/ trimethoprim, and sulfisoxazole on MHII agar. Same

exceptions were observed on BHI agar but with 6mm, 8mm, and 6mm from bacitracin, erythromycin, and penicillin.

E. coli’s major zone range on BHI is 17.5mm from amoxicillin to 27mm from sulfisoxazole. S. aureus on MHII

agar has two lowest zone measurements of 14mm from bacitracin and 14.5mm from streptomycin. All other

antibiotic disks have inhibitory zones of 17.5mm from nalidixic acid to 34.5mm from cefazolin . On BHI, S. aureus

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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.

Physical and Chemical Methods of Control

S. aureus showed little to no change in growth on nutrient agar between the streaked control culture

on section 1 and the streaked culture after a 40ºC water bath on section 2. The only visible difference is that the

control grew slightly thicker lacking the several small, nearly isolated colonies present on section 2. B. cereus

shared the same properties for the streak after an 80ºC water bath on section 3 as S. aureus. The streaked culture

after an 80ºC water bath on section 3 yielded no growth. Only the streak lines are visible on the agar surface. The

streaked control culture on section 1 was also only slightly denser than the streaked culture after a 40ºC water bath

on section 2. This is apparent because of the appearance of a darker colored control culture when the agar is viewed

without the lid from the top and section 1 and section 2 are compared.

The control nutrient agar Y-plate prepared for UV irradiation comparison grew quite well with thick culture

lines grown on and beyond the streak lines on the agar from the inoculating loop. B. cereus has distinct, dense

grown colonies with few visible thin regions or isolated colonies. Like its culture grown after the heat bath, B.

cereus has a creamy to light beige color with a surface that looks softer than E. coli and S. aureus. S. aureus is the

thinnest culture of all and has several places along the streak lines where the agar is visible. The surface also looks

both rigid and rough. E. coli splits the difference with a smooth creamy white to beige surface in some places to a

thin, almost transparent, and rough surface in other places . Whether exposed to UV radiation for 2 minutes or 5

minutes, all three organisms showed no growth on their streaked sections of the nutrient agar Y-plate.

For growth after exposure to a disinfectant, a number was awarded based on amount of growth on a scale

of 0-4. 4 represents unrestricted or heavy growth; it should be identical or close-to-identical to the culture. 0

represents no growth was observed, 1 means growth was severely restricted, 2 means moderate growth restriction,

and 3 means little or slight growth restriction. These growth number assignments are summarized in Table 2.

(Simmons, General Microbiology Laboratory Manual). For comparison, these numbers were lined graph versus

time by each organism in Chart 3, Chart 4, and Chart 5. Only B. cereus showed a reduced growth after exposure to

sterile water. 1% Phenol had no effect on E. coli growth and failed to continue to decrease growth in both S. aureus

and B. cereus after an initial reduction. Betadine, 70% ethanol, 10% bleach, Foam wash with 0.13% Benzalkonium

chloride, 62% hand sanitizer, and 70% isopropyl alcohol all appeared to completely limit growth in all three

organisms. 1% Bleach was able to completely eliminate growth at 30 seconds in both B. cereus and E. coli, and at

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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. (Simmons, General Microbiology Laboratory Manual 73).

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. (Simmons, General Microbiology Laboratory Manual 73).

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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(Roe). 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.

Antibiotic Susceptibility Testing

The Kirby-Bauer technique for antibiotic testing produced similar relative results on both the BHI and

MHII media. E. coli, S. aureus, and P. aeruginosa all have almost identical total zones of inhibition between media;

while E. faecalis, S. typhimurium, and B. subtilis all have large different total zones of inhibition between media (see

Charts 1 and 2). Slight changes did occur in the individual antibiotic zones of inhibition. Bacitracin seemed to have

the larger zones of inhibition around gram-positive bacteria, with the exception of B. subtilis. Bacitracin blocks

bactoprenol recycling and thus cell wall synthesis and overall cell growth. The cell-wall restriction component is

what makes it effective again gram-positive bacteria. (Simmons, An Introduction to Antimicrobials 6). Cefazolin

appeared to drastically cause large zones of inhibition in all organisms tested except the gram-negative P.

aeruginosa. This is because cefazolin is a first generation cephalosporin that acts by blocking cell well synthesis in

much of the same manner as penicillin. Cephalosporins also are effective to a certain degree to gram-positive

organisms depending on the generation of cephalosporins. (Simmons, An Introduction to Antimicrobials 2). The

sulfamethoxazole and trimethoprim disk had large zones of inhibition for all organisms tested, except P. aeruginosa

and E. faecalis on the MHII agar, indicating the organisms are susceptible. When combined, these antibiotics are

called a co-trimoxazole and inhibit the synthesis of folic acid and thus the synthesis of thymidine and uridine.

(Simmons, An Introduction to Antimicrobials 6). A limited effect was observed for P. aeruginosa because this

organism has two membrane proteins, OprM and OprJ, which work as multiple drug efflux systems. According to T

Köhler, et al, one of their multidrug efflux systems is mainly responsible for the intrinsic resistance of P. aeruginosa

to TMP[trimethoprim] and SMX[sulfamethoxazole]. (Köhler, Kok and Michea-Hamzehpour 2288). E. faecalis

resistance was most likely measurement error. Erythromycin strictly showed susceptibility to all gram-positive

organisms tested on both BHI and MHII because it is a member of the macrolides. It acts by blocking protein

synthesis with the 23S rRNA. Gram-positive organisms tend to collect more of the drug. (Simmons, An

Introduction to Antimicrobials 3). Penicillin also strictly made the gram-positive organisms tested susceptible.

Penicillin works by inactivating penicillin binding proteins (PBPs) that are often vital to bacteria cells. Penicillin

thus inhibits cross-linking during cell wall synthesis resulting in primarily gram-positive cell lyses. (Simmons, An

Introduction to Antimicrobials 1). Tetracycline inhibited growth from all organisms tested on both BHI and MHII

agar except P. aeruginosa. Tetracyclines prevent protein synthesis by blocking transfer RNA (tRNA) attachment to

the RNA. They are effective against α-hemolytic and γ-hemolytic streptococci and gram negative bacilli.

Streptomycin was effective against all organisms tested on both media except for E. faecalis with P. aeruginosa

showing moderate susceptibility. Streptomycin is an aminoglycoside that inhibits protein synthesis and is mainly

effective against most gram-positive, gram-negative, and mycobacteria. (Simmons, An Introduction to

Antimicrobials 2). Kanamycin is a member of the same antibiotic family and is primarily effective against gram-

negative bacilli. It is ineffective against the Pseudomonas species, explaining why P. aeruginosa was the only

tested organisms not susceptible. (Simmons, An Introduction to Antimicrobials 2). Sulfisoxazole is the sulfamide

component of the co-trimoxazole discussed earlier. Its role is inhibiting dihydropteroate synthase which in turn

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inhibits the first step of folic acid synthesis. The, like cefazolin, prevents thymine and uridine synthesis. Only E.

coli, S. aureus, and B. subtilis were susceptible. Sulfisoxazole affects a wide variety of gram-positive and gram-

negative bacteria. (Simmons, An Introduction to Antimicrobials 5). It was concluded that S. typhimurium and E.

faecalis were both not susceptible because they must bypass the block of dihydropteroate 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. (Simmons, An Introduction to Antimicrobials 6). 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.

Physical and Chemical Methods of Control

Both B. cereus and S. aureus had similar amount of growth for the inoculated control on section 1 and the

inoculated culture after a 40ºC water bath on section 2. This indicates that 40ºC is not enough of a temperature

increase to restrict growth of S. aureus and B. cereus. This is because both organisms grow at their optimum at

37ºC. For both organisms, no growth was observed on section 3 containing the culture after an 80ºC water bath.

This temperature is much higher than the organisms’ comfortable growing environment. It was noted that B. cereus

should have had a small amount of growth because it is known to be spore forming. This would allow the bacterium

to escape the harsh conditions as spores, and then germinate when conditions improved. This was not observed.

Different areas of the broth in which the sample was taken might have had varying temperatures. The sample might

have come from a portion that had a temperature to severe for spore survival.

UV data was observed as no growth across the board. This was because of the high sensitivity of bacterial

DNA to ultraviolet light. The light waves cause thymine dimers to form and this leads to the death of bacterial cells.

(Kennell)

Testing B. cereus, S. aureus, and E. coli with various disinfectants provided interesting trends (see Charts

3, 4, and 5). 1:100 Dilution of Hand soup was observed to be an overall poor growth restrictor in all organisms

despite length of exposure. Neosporin with 400 units of bacitracin, 3.5mg of neomycin, and 5 units of polymycin B

showed little effectiveness. Ultimately it appeared to come down to a liquidity factor. For example, the foam wash

and antiseptic wash both had the same 0.13% Benzalkonium chloride active ingredient. The antiseptic wash, the

one easier mixed with the specimen sample, caused significantly better restriction. 1% Phenol was also showed to

be a relatively pore disinfectant. This might have been because of the low concentration.

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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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