Grzegorz Koziel

BIO 210
6/2/17
Identification of Unknown #56 – Lab Report

Background information:

Over the years, the importance of cell phones in modern society increased dramatically. What

was once a device strictly for communication became a powerful tool for research, data storage, and

entertainment. Cell phones are used in private as well as in professional environment. They are usually

stored in pockets, handbags, and are often in contact with various surfaces throughout a day, including

user’s skin. A person owning a cell phone touches it even few hundred times a day which makes it a perfect

environment for bacteria to spread. In 2015, at University of Port Harcourt Teaching Hospital in Nigeria,

researchers examined mobile phones of medical personnel for the presence of bacteria. The study

concluded that, “The percentage occurrences of isolated bacteria from the mobile phones of medical

personnel were: Coagulase negative Staphylococci 43.8%, Staphylococcus aureus 25.6%, Streptococci

17.7%, Pseudomonas 7.4% and E. coli 5.3% respectively” (Amala, Ejikema, 2015). Another study conducted

on health care workers in 2009 found, “In total, 94.5% of phones demonstrated evidence of bacterial

contamination with different types of bacteria. The gram-negative strains were isolated from mobile

phones of 31.3% and the ceftazidime resistant strains from the hands were 39.5%. S. aureus strains

isolated from mobile phones of 52% and those strains isolated from hands of 37.7% were methicillin

resistant.” (Ulger, Fatma, Esen, Dilek, Yanik, Gunaydin, Leblebicioglu, 2009). In both cases, the most

common bacteria found on the cell phones were Coagulase negative Staphylococci (CoNS), Staphylococcus

areus, and Streptococci. These bacteria cause a range of problems such as skin infections, food poisoning,

and respiratory infections. They are also very likely to develop antibiotic resistance due to high frequency

of cross-contamination.
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Antibiotic resistance is defined as, “ability of microbes, especially certain bacteria, to overcome

substances that might otherwise kill them or interfere with their growth” (Funk & Wagnalls New World

Encyclopedia, 2016). Bacteria can reproduce at a rapid rate which accounts for its ability to mutate. As

the bacteria spread and cross contaminates, its genome can mutate and develop an antibiotic resistance.

There are couple ways in which bacteria can resist a drug. It can do it by developing specialized proteins

that block the entry of antibiotics, using enzymes to change the shape and function of the antibiotic, using

efflux pump that pushes the antibiotic out of the cell, and developing an enzyme that breaks down the

antibiotic. When penicillin was invented in 1928, many of bacteria caused disease could be easily cured.

However, as the bacteria mutated and acquired antibiotic resistance, it became harder to target them.

Also, it takes much longer for new antibiotics to be developed than it used to. Antibiotic resistance in

bacteria is becoming a serious issue because it is hard to invent new drugs and the globalization allows

various bacteria to cross mutate, creating new types of bacteria.

To develop an effective antibiotic, it is important to identify the bacteria and target specific parts

of it. One of the fundamental techniques used to identify bacteria is a gram stain. This procedure targets

the peptidoglycan of bacteria and allows to determine whether its gram positive or gram negative as well

as helps in identifying the shape of it. Other tests used to identify bacteria are acid-fast stain, growing

bacteria on selective and differential media, catalase test, motility test, antibiotic resistance, etc. All those

tests are used to determine the structural and functional attributes of the species. One of the most useful

tools in identifying species is the Bergey’s Manual established in 1936, “The identification schemes

of Bergey's Manual are based on morphology (e.g., coccus, bacillus), staining (gram-positive or negative),

cell wall composition (e.g., presence or absence of peptidoglycan), oxygen requirements (e.g., aerobic,

facultatively anaerobic) and biochemical tests (e.g., which sugars are aerobically metabolized or

fermented)” (Science jrank.org, 2016).

 Grzegorz Koziel
BIO 210
6/2/17
During the first lab of microbiology, the class collected a sample from their private cell phones

and transferred it into agar plates to observe the growth. The article titled, “Are we aware how

contaminated our mobile phones with nosocomial pathogens?” touched on a topic of bacteria

contaminating private phones in medical environments. It is important to understand that hygiene is

important not only in private life but also in many professions. However, many people disregard the fact

that cell phones might be the source of many bacterial diseases because they are constantly in contact

with skin, mouth, and other surfaces. As the class discovered, many of the private cell phones were full of

different bacteria and molds. Throughout the course, the class learned how to identify different types of

bacteria by using various staining procedures, growing it on differential and selective plates, and other

tests. By identifying certain features, the class learned how to recognize different bacteria. It was a great

learning experience that helped to understand that bacteria can easily transfer between different media,

including cell phones, which in some circumstances, might be very dangerous.

Methods:

To identify the species of bacteria, number of tests were conducted on the unknown #56. It was

tested for morphology and chemical characteristics. The procedure for each test is described below.

I. Isolation of a pure culture

A streak plate was used to isolate a pure culture of bacteria. To prepare a streak plate, the bottom

of the agar plate was labeled. Then, the loop was used to transfer bacteria from the sample to quadrant

1. The loop was used again to gently strike over the surface in a zigzag fashion until the quadrant was

covered. Then, the loop was flamed and cooled down. The loop was drawn across quadrant 1 to pick up

some bacteria and continued to strike in a zigzag across quadrant 2. The process was repeated for

quadrant 3 and 4, beginning the strike at a previous quadrant. The loop was flamed and cooled between

each strike.
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6/2/17
II. Use of selective and differential media

Selective and differential media were used to identify the characteristics of unknown #56. The

blood agar (BA) and Mannitol Salt Agar (MSA) were used because unlike other media they allow gram-

positive bacteria to grow on it. To prepare the plate, a sterilized inoculate loop was used to streak the

bacteria across the plate in a Z pattern. A thin film of bacteria on the plate was be observed. Then, the

plates were incubated for 24 hours at 37 °C.

Another type of differential process used Triple Sugar Iron (TSI) media. To prepare the agar, a

sterilized and cooled inoculation needle was used to select a colony from the unknown streak plate, and

then stabbed into the agar. Then, the loop was used to streak the surface. Plates were incubated for 24

hours at 37 °C.

III. Motility Agar

Motility agars are used to identify whether the bacteria can move. It is thick enough to prevent

movement yet fluid enough to allow bacteria with flagella to move. The agar was prepared by using a

sterilized inoculated needle and stabbing to the center of the tub once. Then, it was incubated overnight.

IV. Catalase Test

To perform a catalase test, a sterilized inoculate loop was used to spread the bacteria onto a clean

microscope slide. Then, a drop of hydrogen peroxide was added and observed. Positive reaction resulted

in forming of bubbles, while negative reaction didn’t form any bubbles.

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V. Optimal Growth Temperature of Bacteria

To determine the optimal growth temperature of bacteria, a nutrient agar was divided into four

segments. Each person had one segment where the unknown bacteria was inoculated with a sterilized

loop in a zigzag fashion. For each unknown, three agar plates were prepared and incubated for 24-48

hours at different temperatures: 25 °C, 30 °C, and 37°C.

VI. Gram Stain

Gram staining allowed to determine the shape of the bacteria and whether it is gram-positive or

gram-negative. To perform a gram stain, a smear was prepared. The loop was used to transfer bacteria to

a clear slide and allowed to air dry. Then, the bacteria were heat-fixed to the slide by running it over the

flame 2-3 times. Once the bacteria smear was ready, the staining procedure began by treating the sample

with the primary stain, crystal violet. Then, it was followed with an iodine solution, a mordant that helps

the stain bind to bacteria. Then, a decolorizer such as 95% ethanol was used. Finally, a counterstain such

as safranin was applied. In each step, it was important to allow 30 to 60 seconds for the treatment to take

place. At the end, the stain was gently washed with water. The slide was observed under a microscope at

100x magnification. Gram-negative bacteria would appear pink while the gram-positive bacteria would

look purple.

VII. Antibiotic Resistance

The antibiotic resistance tested the ability of the bacteria to grow in the presence of five antibiotic:

Ampicillin, Erythromycin, Tetracycline, Bacitracin, and Chloramphenicol. The nutrient agar was divided

into six sections, one for each antibiotic and one for control group. Then, 1-2 colonies of the unknown

bacteria were transferred to a nutrient broth and mixed together. Next, the pipette was used to transfer

0.2 mL from the broth with bacteria culture into the previously labeled agar plate and spread across with
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sterile swap. Finally, the antibiotic disks were transferred to the plate with sterilized forceps and lightly

pressed into the agar. The agar was then incubated at 37 °C. The zone of inhibition was used to determine

whether the bacteria is antibiotic resistant to previously listed antibiotics.

Results:

Figure 1. Streak Plate – Unknown #56

The above streak plate for unknown #56 was a success as some individual colonies were observed in

quadrant 3 and 4. However, there was some contamination in quadrant 1-3 that could be caused by

improper handling of the plate and seen as big white dots.

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Figure 2. Gram Stain – Unknown #56

Gram staining allowed to determine the shape of the bacteria and whether it is gram-positive or gram-

negative. Unknown #56 was determined to be a gram-positive coccus.

 Grzegorz Koziel
BIO 210
6/2/17
Figure 3. Catalase Test

The catalase test for unknown #56 was positive. It was indicated by forming of bubbles after the hydrogen

peroxide was added to the unknown. Presence of catalase meant that the unknown microorganisms can

protect itself from toxic forms of oxygen in nature.

Figure 4. Blood Agar

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Blood agar (BA) is a non-selective differential medium. It was used to distinguish between hemolytic

activities demonstrated by different bacteria. After unknown #56 grew on the plate, it appeared

white/clear. The clear zone around culture zone indicated the complete lysis of the red blood cells.

Therefore, the unknown #56 was determined to be Beta-hemolytic.

Figure 5. Mannitol Salt agar

Mannitol Salt agar (MSA) is a selective and differential medium. When the unknown #56 was tested, the

bacteria appeared clear. The absence of growth indicated that the bacteria is not a staphylococcus.
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Figure 6. Temperature plate at 25°C

The unknown #56 grew a little at the 25°C. However, the plate was left at room temperature for too long

before it was put in the refrigerator. The small amount of growth happened at the room temperature and

once it was put in the freezer, the growth stopped. Therefore, it was assumed that the growth was

insignificant and was caused by an experimenting error.

Figure 7. Temperature plate at 30°C

The bacteria from unknown #56 did not grow at all at 30°C.
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6/2/17

Figure 8. Temperature plate at 37°C

The growth of bacteria #56 at 37°C was significant. In fact, it seemed to grow very well relatively to other

bacteria and different temperatures. Therefore, it can be assumed that 37°C is the optimal temperature

for its growth and that unknown #56 is a Mesophile.

Figure 9. Antibiotic Plate

 Grzegorz Koziel
BIO 210
6/2/17
The inhibition zone was measured in diameter around each antibiotic. Then, it was compared with the

table in lab manual to indicate whether the bacteria are sensitive or resistant to an antibiotic.

Table 1. Antibiotic resistance

Antibiotic Diameter Sensitive or Resistant?

Ampicillin 17 mm Sensitive

Erythromycin 30 mm Sensitive

Tetracycline 8 mm Resistant

Bacitracin 38 mm Sensitive

Chloramphenicol 20 mm Sensitive

Figure 10. Citrate, Motility, and TSI tests

 Grzegorz Koziel
BIO 210
6/2/17
The citrate test of unknown #56 indicated negative results as the color of the media did not change.

Therefore, the bacteria can’t use citrate. The motility test was also negative because the bacteria grew

only in the area that was pierced and did not move through the media. The triple sugar iron (TSI) test was

negative as well. It was indicated by the absence of black iron sulfide precipitates.

Discussion:

The first experiment, the streak plate, was very useful in isolating colonies. The plate was also

used in further tests as the source of the sample. The next tests, gram staining, was perhaps the most

important one. When researcher attempts to identify bacteria, the first thing to look at is whether the

unknown is gram-positive, gram-negative, or gram-variable. It also allows to see the morphology of the

sample. The unknown #56 was determined to be gram-positive which determined what kind of tests were

conducted next and greatly narrowed down the identification of the bacteria. The catalase test is useful

in determining whether the bacteria are aerobes or anaerobes. Also, “the catalase test is primarily used

to distinguish among Gram-positive cocci: members of the genus Staphylococcus are catalase-positive,

and members of the genera Streptococcus and Enterococcus are catalase-negative (Acharya, 2017). As the

unknown #56 tested positive for presence of that enzyme, it can be assumed that it is not a Streptococcus.

As the unknown was earlier determined to be gram-positive, a Mannitol Salt Agar (MSA) and Blood Agar

(BA) tests were conducted. The BA test showed that unknown #56 was beta-hemolytic. The MSA was

significant because it showed no growth which proves that the bacteria are not a Staphylococcus. The

temperature plates showed that unknown #56 is a mesophyll because it grew on 37 °C plate. The antibiotic

test indicated that the bacteria is resistant to Tetracycline. The motility test was negative, which shows

that the bacteria is missing flagella. TSI and citrate tests were also negative.

During the course of the experiment, the only problem encountered was the contamination of

the streak plate. The molds present on the plate could interfere with other tests that used the material
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6/2/17
from the streak plate. To avoid mistakes like that, it is important to maintain clean work station and cover

the agar plates when they are not in use. Otherwise, the techniques used in lab were efficient. They were

simple but still allowed to determine many characteristics of the bacteria.

Figure 11. Dichotomous tree

Gram +

Cocci Bacilli

Mannitol No mannitol
Streptomycetes Staphylococcus
Fermentation fermentation
Streptomyces
Staphylococcus Bacillus megaterium
griseus
epidermidis Bacillus subtilis

Motile Nonmotile
Streptococcus Micrococcus
Bacillus cereus Corynebacterium
pseudodiphtheriticum
Micrococcus
luteus

No growth at Growth at
10°C 10°C
Streptococcus
lactis
No mannitol Mannitol
fermentation fermantation
Streptococcus Streptococcus
salivarius
mutans

The dichotomous tree created was used to identify the unknown #56. The gram stain showed that

the unknown bacteria was a coccus which greatly limited the possibilities. Furthermore, the catalase and

MSA tests indicated that the unknown is not a Staphylococcus or Streptococcus. In this case, the only 2

choices left were Streptomyces griseus and Micrococcus luteus. Streptomyces are characterized by

filamentous shape which was not observed during the gram stain. Therefore, the unknown #56 was

determined to be Micrococcus luteus.

 Grzegorz Koziel
BIO 210
6/2/17
According to the eol.com, “Micrococcus luteus is a Gram-positive, to Gram-variable,

nonmotile, Coccus, saprotrophic bacterium that belongs to the family Micrococcaceae. It is urease and

catalase positive. An obligate aerobe, M. luteus is found in soil, dust, water and air, and as part of the

normal flora of the mammalian skin” (Encyclopedia of Life). The motility test which was negative, along

with the fact that is sensitive to bacitracin, increases the certainty that the unknown #56 is Micrococcus

luteus. Another test that could be used to ensure it could be the nitrate reduction test, “Nitrate broth is

used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the

enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and

nitrite to produce molecular nitrogen” (Micrbugz.com). In the case of Micrococcus luteus, the nitrate test

should be positive.
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6/2/17
References

Acharya, Tankeshwar. "Catalase test: principle, uses, procedure and results." Microbeonline. N.p., 09
May 2017. Web. 02 June 2017.

Amala, S. E. and I. F. Ejikema. "Bacteria Associated with the Mobile Phones of Medical
Personnel." American Journal of Biomedical Sciences, vol. 7, no. 1, Jan. 2015, pp. 26-32.

"Antibiotic Resistance." Funk & Wagnalls New World Encyclopedia, 2016, p. 1p. 1.

"Bacteria - Identifying And Classifying Bacteria." Gram, Stain, Dna, and Bacterium - JRank Articles.
Science Jrank, n.d. Web. 02 June 2017.

"Micrococcus luteus - Overview." Encyclopedia of Life. N.p., n.d. Web. 02 June 2017.

"Nitrate Reduction Test." Welcome to Microbugz - Nitrate Reduction Test. N.p., n.d. Web. 02 June
2017.

Ulger, Fatma, Saban Esen, Ahmet Dilek, Keramettin Yanik, Murat Gunaydin, and Hakan Leblebicioglu.
"Are we aware how contaminated our mobile phones with nosocomial pathogens?" Annals of Clinical
Microbiology and Antimicrobials. BioMed Central, 06 Mar. 2009. Web. 02 June 2017.