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The Sampling and Culturing of Environmental Microbes and

Prepared Bacteria for the Understanding of Basic Microbiology Lab

Techniques, Identification of Various Bacterial Morphologies, and

Analysis of Bacterial Growth

Michael Wallach

INTRODUCTION

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In all imaginable environments humans encounter, microorganisms surround and thrive.

Whether they survive through anaerobic or aerobic processes, extreme cold temperatures, or high

pressure environments, their vast diversity is easily shown marking their avoidance as

impossible. Microbes have been able to evolve overtime to reveal a wide spectrum of microbial

diversity today. In this lab, the constant, unavoidable encounter with numerous types of

microbes was illustrated most effectively in studying morphological details of colony growth and

cell shapes from samples taken from several every day locations. The practice with and

understanding of various techniques was performed through use of these microbes obtained as

well as through prepared Escherichia coli and Staphylococcus aureus cultures. Through the

technique of streak plating, we learned how to grow bacterial cultures on various types of agar

media and how to isolate individual colonies for further study. Several of these media types can

be used to select or deselect for growth of one type of microbe over another, show through

various color changes a microbe’s enzymatic activities, or simply promote growth of many types

of microbes. Furthermore, the importance of aseptic techniques was shown and how simple

steps, such as heating an inoculating loop, can reduce unintended bacterial growth caused by

contamination of the surrounding environment. We learned how to identify a colony’s

morphological features through their color and a classification of various common shapes. These

classifications were broken down into 3 categories: form, elevation, and margin (see figure 1).

The technique of gram staining promoted the visibility of bacterial cell structures and aided in

Figure 1: Different Forms, Elevations, and Margins in Bacterial Colony Morphology (Kennell 39, in Nutrition, Culturing, and Growth)

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the understanding of cell wall characteristics. Gram-positive cells have a cell wall surrounding

the cell membrane composed of a thick, peptidoglycan layer that is not readily diffusible by

crystal violet dye complexes (Kennell 20, in Lab Manual). These complexes are therefore more

difficult to wash off of the cells when membranes are fixed with iodine than in gram-negative

cells. In these cells, the cell wall is composed of a thin peptidoglycan layer located interior to an

outer, easily permeable cell plasma membrane. This collective cell wall is also located exterior

to the inner cell plasma membrane. Because of the peptidoglycan’s thin structure combined with

the outer membrane’s permeability, the crystal violet complexes are allowed to easily diffuse

when the cell is washed with a polar solvent (Lab 20). Safranin red is added last typically to

show a contrasting pink color of the gram-negative cells with the darker purple color of the

gram-positive cells. The techniques of a negative stain where also utilized and it was shown to

be beneficial in cells that are composed of an outer capsule that prevents entry of various dyes.

The result is a stained background with unstained bacterial cells located throughout. The cells

further resist the dye because they have negative outer charges that repel the negative charges of

the acidic dyes (Lab 21-22). The cells can then be classified based on individual cell shape

(spherical/cocci, rod-like/bacilli, or helical/spirilla) and in the groups the individual cells form.

For example, cocci cells can either be diplococci (attached in pairs), streptococci (attached in

chains), tetrads (groups of four cells), sarcinae (in a cuboidal arrangement), or staphylococci

(attached in clusters). While rod-like or bacilli cells are usually found as single cells but can

sometimes attach in pairs (diplobacilli) or chains (streptobacilli). Through spectrophotometry we

learned how light absorbance increases can be detected throughout time as a reflection of

bacterial colony growth in liquid culture and how this data can be used to produce a growth

curve. With pour plating, we learned how to calculate a liquid culture’s original colony density

based upon a series of diluted samples. Perhaps most importantly, it was shown how mistakes

when well documented and reflected on, can be a crucial method to learn from.

MATERIALS AND METHODS

First, eight samples were obtained from the surface of four different outdoor

environments: tree bark, grass blade, open public ashtray, and bench tabletop. One sample was

collected from two different sites from each environment. To obtain these samples, a sterile

cotton swab was first moistened with distilled (DI) water. Then, an area of approximately 2-3

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inches was rubbed completely while moving the cotton swab in a rolling fashion. Next, the

sample was streaked onto an agar growth plate by lightly rubbing the cotton swab back and forth

in the same rolling fashion onto the agar. The streaking was repeated with the same cotton swab

after rotating the agar plate approximately 90º. One sterile cotton swab was used for each

sample (8 total). Four different solid growth mediums were used: two nutrient agar plates, used

for the general cultivation of bacteria; two brain-heart infusion agar plates (or BHI), used in the

cultivation of aerobic and anaerobic microorganisms; two saboraud dextrose agar (SD) plates,

used for the culturing of fungi-like yeasts and molds; and two trypticase soy agar (or TSA) with

5% sheep blood, used for the isolation, cultivation, and detection of hemolytic activity of

microorganisms (Lab 9). Each sample site was streaked onto one agar plate and labeled with all

group member’s initials, date, sample and site, and lab section. The sites and their respective

agar mediums they were streaked onto are summarized in Table 1. All plates were inverted and

incubated at 30ºC for 48 hours and observations regarding growth were taken for the site with

most growth from each environment. Further, one plate from another group showing growth

from a sample taken from a human left foot on BHI agar was also observed. It should be noted

that this plate was inverted and incubated at 37ºC, the standard temperature used for inoculation

of human body samples.

Table 1: Respective Media Used for Samples Obtained Sites from Various Environments

Sampled EnvironmentNutrient

AgarBHI Agar

Saboraud Dextrose

Agar

TSA with 5% sheep

blood

AshtraySite A XSite B X

Bench TableSite A XSite B X

Tree BarkSite A XSite B X

Blade of Grass

Site A XSite B X

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After the incubation period, one colony from site A of the bench table and one colony

from site A of the tree bark were both streaked for isolation of colonies onto separate BHI agar

plates and allowed to incubate inverted at 30ºC. To streak for isolation, a wire inoculating loop

was used to streak the colony onto the agar plate in a back and forth motion at the top of one

side. The plate was then turned approximately 45º and streaked again in the same fashion,

making sure to only cross the previous streak a few times. This process was repeated two

Figure 2: Streak Plate Technique (Nutrition 33)

additional times so that four total streaks result (see figure 2). Aseptic technique was also used

throughout this process, flaming the inoculating loop for several seconds, allowing it to turn red,

before and after contact with any surface. The loop was allowed to air cool to room temperature

before contact with a specimen and an agar plate. This prevented contamination from other

microbes. A bacterial broth mixture containing S. aureus and E. coli was streaked for isolation

on a nutrient agar and incubated inverted at 37ºC. In this case to obtain the specimen, a liquid

broth culture was used. The wire loop, after cooling, was dipped into the broth vial before the

initial streaking. Next, a pure broth culture of E. coli was streaked for isolation onto four

lysogeny broth (or LB) plates. LB plates are a generic rich media suitable for growing many

aero-tolerant species of bacteria, including E. coli, Bacillus subtilius, S. aureus, or

Staphylococcus epidermidis and different yeasts including Saccharomyces and Candida species

(Pouring LB Agar Plates 1). To test the effects of temperature on E. coli growth, each LB plate

was inverted and incubated at 4ºC, 23 ºC, 37 ºC, and 55ºC respectfully for 120 hours.

Observations for all 6 plates were taken.

After the incubation period, two bacterial smears were prepared from an isolated colony

of E. coli from the LB plate at 37ºC and S. aureus from the mixed nutrient plate. To prepare a

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bacterial smear, an inoculated wire loop was dipped into a bottle of DI water to transfer a drop to

a new blank glass slide. Next, the isolated colony was transferred with the loop to the slide and

swirled around in the water drop in an area of approximately 15 mm. After allowed to air-dry,

the smear was then heat fixed by waving the slide three times through a Bunsen burner. Aseptic

techniques were used as described above. A gram stain was then performed on both cooled and

air-dried smears. This was accomplished by completing a standard four step process: cover the

smear with crystal violet for one minute and rinse with water, cover the smear with iodine for

one minute and wash off with an acetone/ alcohol mixture, and cover the smear with Safranin red

for one minute and rinse with water (Lab 21). A coverslip was added after the slide was air-

dried. Another bacterial smear was created using a tooth pick to obtain a sample orally between

a tooth and the gum line. This smear was fixed and only stained with crystal violet (step one of

the gram stain technique). Smears and gram stains were also performed from colonies for the

bark site A and bench table site A from the isolation attempts described earlier. A negative stain

was performed on a prepared broth culture of Bacillus cereus. This was accomplished by first

adding a drop of the negative stain nigrosin. Next, the bacterial sample was added to the stain

using a wire loop and mixed. A second slide was placed on top of the sample containing slide

allowing the stain to naturally spread out. The slides were then taken apart and a coverslip was

added once allowed to air-dry (Lab 22). Aseptic techniques were used in all smear preparations.

Lastly for comparison, smears from the Carolina number 29-3946 slide were viewed under 400x

and 1000x magnification. The 1000x, or oil immersion, magnification was performed by placing

a drop of oil on top of the cover slip while in between the 40x and 100x objectives on a

compound light microscope. The 100x objective was then moved into position and focused.

This was done after first focusing the stained smear under all three other objectives stepwise (4x,

10x, 40x). Observations for all smears were recorded when viewed under the oil immersion

objective.

To determine the rate of E. coli growth, Spectrophotometric readings were taken and

recorded as a class at various times from 9:30AM – 4:30AM of an inoculated flask containing

2mL of the sample. Each group recorded two readings, first zeroing the spectrophotometer at a

600nm wavelength with a 2mL flask of medium (Lab 31). For each reading, the inoculated flask

was inserted into the spectrophotometer and, after a few seconds, the absorbance units displayed

was given to the lab section’s teaching assistants to include in a collective table. Sample

spectrophotometric data for E. coli was also given from various periods from 8:00AM – 4:50PM

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for comparison (shown in table 3 of results) (Lab 30). The pour plate method was also

performed by each group to obtain a set of five dilution plates from 10-3 – 10-7. This was

accomplished by first transferring 1 mL of E. coli broth to a test tube containing 9mL of saline

using a 1mL pipette. This was labeled tube 1 and represented 10-1 dilution which was used to

transfer 1mL into tube 2 (also containing 9mL of saline) with a new 1mL pipette tip. This was

repeated until seven dilutions were obtained (tubes 1-7 corresponding to dilutions 10-1-10-7). All

test tubes, as well as the original sample, were mixed with a vortex prior to transfer. Melted

nutrient agar deeps (labeled 10-3-10-7) were used to mix with 1mL from tubes 3-7 respectfully

with a vortex. The agar deeps were allowed to cool from their original temperature of 80ºC for

no more than five seconds before each dilution tube sample was added. Five nutrient agar plates

all labeled with the standard information and corresponding dilutions, were used to receive the

pour from each agar deep tube (labeled 10-3-10-7). After the five pour plates were cooled to room

temperature, they were inverted and incubated at 37ºC for 48 hours. Colony forming units (cfu)

and observations were recorded for each pour plate.

RESULTS

Sample site B from the ashtray grown on TSA with 5% sheep blood yielded growth of

three visibly different colony types. One colony was flat and large in diameter with a

filamentous overall form and filaform margin. This large colony was also dark green in the

center. The other two colony types observed were uniformly circular and small in diameter and

of equal average size. The first smaller colony displayed no color change and was flat with the

agar surface. The second smaller colony displayed an overall green color and was slightly raised

from the agar surface. Site A from the bench table grown on nutrient agar displayed two visibly

different colonies. The first was entirely round, white in color, and small in diameter. Further, it

was slightly raised from the agar surface. The second colony appeared a couple of orders larger

than the first and was overall filamentous with a filaform margin. However, the center was black

in color and umbonate from the agar surface. The site B sample from the tree bark grown on

blood agar showed growth of one colony type that appeared irregular in form. Its margin was

undulate and was slightly raised from the agar surface. Each colony was dark in the center and

transitioned to a cloudy white color on the circumference. Around each colony appeared a clear

ring in the blood agar. Contamination was also observed in that a blade of grass was found in the

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agar. Lastly, the site A sample taken from the grass yielded growth on the SD agar of one

irregular in form colony type. The plate was widely covered with interconnected colonies that

were long and glossy white in color. The colonies were raised from the agar and had a darker

glossy white center. Samples taken from a left foot and grown on BHI agar from another group

were also observed and showed growth of two colony types. The first was irregularly shaped

with a curled margin and flat rise. Further, this colony displayed a swarming growth pattern.

The second was raised and had circular overall form. Both appear creamy white. A summary of

these results are shown in Table 2.

Table 2: Observations of Cultures Grown from Environmental Samples on Various Types of MediaAshtray Site B Bench Table Site A Tree Bark Site

BGrass Site A Left Foot

Colony TypesType 1 Type II Type III Type I Type II Type I Type I Type I Type II

Agar/ temp. grown

TSA w/ 5% Sheep’s Blood/ 30ºC Nutrient/ 30ºC TSA w/ 5% Sheep’s Blood/ 30ºC

Saboraud Dextrose/ 30ºC

BHI / 37ºC

Form filamentous Circular

Circular circular

Filamentous Irregular Irregular Irregular circular

Elevation flat flat raised raised Umbonate Raised Raised Flat raised

Margin filaform entire entire Entire Filaform Undulate entire Curled Entire

Color Dark green center

No color change

Green White White around black center

White around black center. Clear ring in agar around colony

Interconnect glossy white areas. Dark glossy in center

Creamy white swarming growth pattern

Creamy white

Amount & Size (s, m, l)

1, large 3, Small

3, Small Small medium 15+, medium Many, small-medium

2, medium-large

4, small

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Streaking for isolation using the site A sample from the bench table produced few,

small colonies that were round, flat, and white in appearance when grown on BHI agar (figure

3).

Figure 3: Culture from Bench Table Site A Streaked for Isolation on BHI agar

Figure 4: Culture from Tree Bark Site A Streaked for Isolation on BHI agar

For the site A bark-sampled culture, more growth was observed; however, only one colony was

isolated (figure 4). Streaking for isolation on nutrient agar from the bacterial mixture sample of

E. coli and S. aureus showed two colony morphologies. Both were flat with a circular form and

round edge. One was creamy yellow while the other was creamy white. Both formed large areas

of clusters. Isolation of a couple colonies was observed for both morphologies (see figure 5).

Figure 5: Mixed S. aureus and E. coli culture grown on Nutrient Agar at 37ºC

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The pure E. coli sample grown on LB agar plates showed a range of growth depending on the

incubation temperature. The plate incubated at 4ºC showed no evidence of specimen growth

(figure 6). The plate incubated at 23ºC showed growth of a few white colonies (figure 7) similar

in morphology to those seen on the mixed culture shown in figure 3. The plate incubated at 37ºC

showed great growth of the creamy white colonies (figure 8). Also, streaks were easily visible

and many isolated colonies were obtained. The plate incubated at 55ºC also showed large

growth of the creamy white colonies (figure 9), however, was noticeably thinner than the plate

incubated at 37ºC. No contamination was observed in any plate.

Figure 6: Pure E. coli Culture Grown at 4ºC Figure 7: Pure E. coli Culture Grown at 23ºC

Figure 8: Pure E. coli Culture Grown at 37ºC Figure 9: Pure E. coli Culture Grown at 55ºC

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Gram staining of an E. coli smear from an isolated colony produced light purple, slightly

pink colored specimens when viewed under the oil immersion objective at 1000x magnification.

Further, the cells were rod-shaped and found individually scattered. For gram stained S. aureus

under oil immersion, dark purple and circular cells were observed. These cells also seemed to

group with one another forming chains. Gram staining of the tree bark sample and viewed under

oil immersion showed dark purple cells that were rod-shaped and formed clusters. Conversely,

no specimen was observed when a gram stain was performed on a smear obtained from an

isolated colony grown from the bench table sample. A crystal violet stain of the oral sample

viewed under oil immersion showed various shades of violet. One type of dark violet microbe

appeared round and formed clusters. These clusters were very large containing often 100+ cells.

Further, irregular rod shaped cells were observed. These both appeared dark and light violet

throughout the smear. A negative stain of B. cereus when viewed with the 40x objective showed

a large purple background with white rod-shaped cells. These cells formed clusters in the

foreground that were difficult to distinguish. When Carolina Slide #29-3964 was viewed, three

smears were observed. The first, under the 40x objective, had many rod-shaped cells that

seemed to form chains and clusters. Some of these cells appeared pink and others purple. Under

oil immersion, the second smear had many circular cells grouped in large clusters all appearing

pink. The third smear, also viewed under oil immersion, showed light pink, irregular rod-shaped

cells. These cells were often found individually and in clusters of various sizes. A summary of

these results are in Table 3.

Table 3: Summary of Bacterial and Prepared Smear Stain Results and Cellular Morphology

Oral Sample Carolina Slide #29-3964 E. coli S. aureus Tree Bark Sample

Bench Table Sample

B. cereus

Cell Types Type 1 Type 2 Smear 1 Smear 2 Smear 3

Stain Crystal violet Gram Gram Gram Gram Gram NegativeResult Dark

violetLight & dark violet

Gram-variable

Gram – Gram – Gram – Gram + Gram + No specimen observed

Stained background

Morphology Round Irregular rod-shaped

Rod-shaped

Round Irregular Rod-shaped

Rod-shaped

Round Rod-shaped

N/A Rod-shaped

Grouping clusters Clusters & singular

Chains clusters Clusters & singular

singular chains Clusters N/A Large Clusters

Magnification

1000x 1000x 400x 1000x 1000x 1000x 1000x 1000x All 400x

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Color Observed

Dark violet

Light & Dark violet

pink & purple

pink pink Pink/ light purple

Dark purple

Dark purple

N/A Clear/ white cells

Growth curves were plotted using a semi-log line graph of the given spectrophotometric

data found in Table 4. The base 10 logarithm of the absorbance units was placed on the Y-axis

(Chart 1). The collective spectrophotometric data obtained from all lab class sections of the

inoculated E. coli can be found in Table 5. The semi-log line graph of the data can be found in

Chart 2. Dilution plates of the broth culture of E. coli that yielded less than 30 colonies were

marked as TFTC (too few colonies to count). This was the case for dilutions 10-4 – 10-7. For the

dilution plate of 10-3, 184 cfu where observed. This number was obtained by dividing the agar

plate into four quadrants, counting the number of colonies found in one quadrant, then

multiplying that number by four. Further, this number traces back an original concentration of

1.84×105 cfu/mL. This data can be found in Table 6.

Table 4: Sample Spectrophotometric Data Given for Cultures of E. coli at Various Dilutions

25 Hour Time (Hour:Minutes) Flask 1 (1µL)

Flask 2 (10µL)

Flask 3 (100µL)

Flask 4 (1000µL)

8:00 0 0 0 09:00 0 0 0.012 0.037

10:00 0 0.012 0.012 0.16810:30 0.026 0.013 0.039 0.30710:50 0.012 0.02 0.059 0.43411:10 0 0.024 0.079 0.59811:30 0.022 0.02 0.134 0.811:50 0.006 0.035 0.212 1.0412:10 0.036 0.054 0.295 1.14812:30 0.051 0.081 0.499 1.33612:50 0.029 0.105 0.622 1.47213:10 0.035 0.165 0.827 1.53913:30 0.039 0.236 0.947 1.49413:50 0.053 0.378 1.153 1.77214:10 0.102 0.48 1.297 1.72214:30 0.138 0.673 1.407 1.86114:50 0.188 0.817 1.483 1.915:10 0.299 1.055 1.605 2.02815:30 0.387 1.251 1.649 2.02215:50 0.475 1.258 1.649 2.06116:10 0.583 1.347 1.697 2.1716:30 0.792 1.47 1.713 2.12516:50 0.961 1.507 1.804 2.212

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8:00 AM 10:24 AM 12:48 PM 3:12 PM 5:36 PM1.00

10.00

Chart 1: Growth Curve of E. coli Based on Sample Spectrophotometry Data at Various Concentra-

tions

Flask 1 (1µL)Flask 2 (10µL)Flask 3 (100µL)Flask 4 (1000µL)

Time (hour:minutes)

A60

0nm

(log

10)

Table 5: In-class Spectrophotometric Data Obtained for Cultures of E. coli at Various Dilutions over Time

12 hour Time (hours:minutes) Constant Flask 1 (1 µL) Flask 2 (10 µL) Flask 3 (100 µL) Flask 4 (1000 µL)

9:30 AM 0 0 0 0 010:30 AM 0 -0.004 -0.006 -0.003 0.02511:30 AM 0 -0.001 -0.001 0.004 0.13112:00 PM 0 -0.006 -0.004 0.02 0.25112:30 PM

1:00 PM 0 0.3961:30 PM 0 0.019 0.155 0.512:00 PM2:30 PM 0 0.007 0.045 0.316 0.5843:00 PM 0 0.005 0.087 0.469 0.6213:30 PM 0 0.045 0.167 0.592 0.694:00 PM 0 0.043 0.275 0.638 0.7344:30 PM 0 0.048 0.313 0.724 0.715

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9:30 AM

10:42 AM

11:54 AM

1:06 PM

2:18 PM

3:30 PM

4:42 PM

5:54 PM

1

10

Chart 2: Growth Curve of E. coli Based on In-class Spectrophotometry Data at Various Concentra-

tions

Constant Flask 1 (1 µL)Flask 2 (10 µL)Flask 3 (100 µL)Flask 4 (1000 µL)

Time (hour:minutes)

A60

0nm

(log

10)

Table 6: Dilution Plates of Five Concentrations Created From E. coli Broth Culture and Their Relative Colony Forming Units and Calculated Original Culture Concentration.

Dilution Number of Colonies on Plate Total cfu / mL10-3 184 cfu 1.84×105

10-4 Too few to count -10-5 Too few to count -10-6 Too few to count -10-7 Too few to count -

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DISCUSSION

The sample obtained from site B of the ashtray grown on blood agar showed three

distinct colony types as described in the results. The larger filamentous colony appeared green

indicating α-hemolytic activity. This is caused by a “reduction of hemoglobin in the erythrocytes

to methemoglobin” (D.M. Rollins and S.W. Joseph 1). Because of the filamentous form, this

colony was most likely fungal. The other colony, also green but small and round, also had α-

hemolytic activity, however, was most likely bacterial due to its small and entire circular form.

The last colony that displayed no color change was γ-hemolytic. In other words, these colonies

displaced no hemolytic activity. Further, these colonies were also small and with an entire

circular form. This again indicates bacterial growth. The bench table site A filamentous colony

was most likely fungal. This is further supported by the lack of a specimen reported when the

colony was smeared and gram stained after having been isolated. This occurred because fungal

cells lack the peptidoglycan layer necessary to trap the crystal dyes. The other smaller, round

colonies were most likely bacterial. Perhaps the most interesting culture was the appearance of

clear circular rings on the bark site B grown on blood agar. This indicates ß-hemolytic activity.

This is caused by complete hemolysis and is accompanied with a clear zone around colonies on

blood agar medium” (D.M. Rollins and S.W. Joseph 1). These colonies were most likely

bacterial pathogens. For this reason the agar plate had to be sealed off prohibiting any further

colony tests. It would have been interesting to examine the morphologies of the bacterial cells

and the effect of the grass blade contamination. Overall a common trend held was that small,

creamy white, and round colonies were most likely bacterial while the filamentous and filaform

colonies were most likely fungal. Further, the TSA blood agar seemed to induce the most

significant growth regardless of hemolytic activity. The tree bark also seemed to not only host

the greatest diversity of colonial growth but magnitude as well.

When attempting to streak for isolation for both the S. aureus/ E. coli mixture and the

pure E. coli sample, the cultures after incubation clearly showed when compared to the

temperature growth agars that the small, yellow round colonies were S. aureus and the small

white and round colonies were E. coli. The first examination of incubated cultures streaked for

isolation provided limited isolated copies. This was determined, through technique adjustments,

to be caused by streaking too many lines across the previous streak between streak three and

four. This alteration allowed for a better thinning of colonies transiently from streak one to

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streak four. Also, reducing the inoculating amount transferred with the wire loop also aided the

isolation process. However it was still easily visible, as previously shown in Figure 8, that at

37ºC, E. coli experiences its best growth. This is most likely because 37ºC is within the cardinal

temperature range for a bacterial specimen commonly found within the human body. Therefore,

E. coli most likely thrives in warm, damp conditions. At 55ºC E. coli is able to still grow quite

effectively, but just not as fast and as dense as at 37ºC. Furthermore, no growth observed for the

culture incubated at 4ºC for 120 hours indicates that growth must be either completely prevented

or severely limited.

The gram staining results were much of what was to be expected for the E. coli and S.

aureus smears, showing them as gram-negative and gram-positive respectfully. E. coli was

characterized as singular-bacillus and S. aureus as streptococcus. This was incorrect; however,

as S. aureus should have been characterized staphylococcus, forming clusters rather than chains.

Strangely observed was the first smear on Carolina slide number 29-3964. Although it appeared

streptobacilli, the gram stain was difficult to determine containing regions of both very light pink

and of very dark purple. It would be interesting practice to further classify this unknown

organism with other methods. In addition, a gram stain of the oral sample might provide

additional information of interest.

Regarding the spectrophotometer, the data obtained seem extremely variable. This was

best displayed when trying to obtain an accurate absorbance reading for the culture at any given

time. In just a matter of seconds, the reading can fluctuate several digits as far as the tenths

place. This leads to speculation regarding the accuracy of the results. Perhaps it would have

been beneficial to adjust the spectrophotometer to 0 with the control tube between every

measurement, rather than between group turns. Also, the experiment might be more accurate if it

were to be performed several times then averaged before plotted into a growth curve. These

recommendations derive out of the stark contrast between the sample data growth curve (Chart

1) and the class data growth curve (Chart 2). The sample data has very distinct log and

stationary phases, while the class data, although visible, displays very choppy data points.

Furthermore, the semi-log graph’s visual results might be better if the curves were to be

normalized, for example to begin at 0.01 at cycle 2 for Chart 1. This would avoid the

impossibility of the 0 absorbance unit readings and provide a more fluent graph. Furthermore

when performing the pour plating, the E. coli. samples were added to the agar deeps at high

temperatures (only cooling for a few seconds from an initial temperature of 80ºC). This resulted

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in only one plate with cfu’s able to be counted when the results should have produced plates with

too many colonies to be counted (or TMTC). The agar deeps should have been allowed to cool

to around 65ºC before the E. coli dilution sample was added. Not allowing this resulted in the

low calculated original culture concentration found in Table 6.

The experiment was more than effective at teaching how to perform various techniques

of basic microbiology in addition to understanding why they are performed. Colony and cellular

morphology was better understood. In addition, techniques such as plate streaking for isolation

and pour plating were better understood. This lab worked effectively to increase the basic

fundamental understanding and knowledge of a few techniques and elements involved in a

microbiology lab.

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REFERENCES

Kennell, Jack. 2009. General Microbiology Laboratory Manual. Saint Louis University: General

Microbiology 465-37. 9-31.

Kennell, Jack. 2009. Nutrition, Culturing, and Growth. Lecture Powerpoint. Saint Louis University: General Microbiology 464-36.

Pouring LB Agar Plates. 2001. Microbiology Techniques. 1.

http://www.madsci.org/~lynn/micro/techniques/pour_plates/LBagar.html.

Rollins, D.M. and S.W. Joseph. 2000. Basic Definitions. University of Maryland: Pathogenic

Microbiology. 1. http://www.life.umd.edu/classroom/bsci424/Definitions.htm.