Examination of Pathogenic Vibrio from the Ala Wai and their Growth

Daniel HongMichael Zobian

AP BiologyMr. John Kay

Abstract:Water samples were taken from the Ala Wai canal and examined for pathogenic bacteria.

The bacteria are specifically from the genera Vibrio, isolated using TCBS agar plates. The general size of the bacteria was examined after a specific incubation period. Two sample sets were taken, and it was found that sample set I and sample set II both contained varying amounts of growth. Although there is not enough data to support this claim, our data shows more pathogenic Vibro are present near the bridges at a lower tide.

I. Introduction & Research

Bacteria from the genera Vibrio are gram-negative and highly halophilic. The word

halophilic is derived from Greek word meaning ‘salt-loving.’ The halophilic bacteria are

extremophiles that thrive in harsh conditions, specifically environments where salt

concentrations are very high (MicrobeWiki, 2010). These bacteria can cause life threatening

conditions and diseases if an infection is left untreated. This is especially the case, because

vibrios are gram-negative. Beside the ability of gram-positive bacteria to retain a crystal violet

dye in a gram staining protocol as opposed to gram-negative bacteria, gram-negative bacteria

have one distinctive feature. This critical difference is located in the outer membrane of the

bacteria. Gram-negative bacteria have a unique outer membrane, in that it does not contain

certain drugs and antibiotics that protrude from the cell, which is the reason why vibrio are

generally more resistant to antibiotics and are potentially more dangerous than its counterpart

(Kaplan, 2000).

A different component of gram-negative bacteria that contribute to their pathogenic

capability are lipopolysaccharides. Also known as lipoglycans, these lipid/polysaccharide

hybrids are joined by covalent bonds that strengthen the structure of the bacteria, helping to

protect the bacteria from other chemicals. Lipopolysacchardies act as endotoxins, which are

recognized by the human immune system and promote inflammation (Stewart, 2006).

Vibrio can be rod-shaped, straight, or curved. They are motile, and generally move using

a polar flagellum. They can perform respiration as well as fermentation in order to survive. They

are chemoheterotrophs—they obtain nutrients from other plants and animals to produce energy

for work. In reproduction, vibrio divide asexually through binary fission. Vibrio are commonly

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found in marine or estuarine environments—that is, in seawater or a coastal waters. However,

vibrio can also be found to survive in freshwater as well (MicrboeWiki, 2010).

Furthermore, most species of Vibrio are pathogens to human beings. Common species of

Vibrio include V. cholerae, V. parahaemolyticus, and V. vulnificus. These can result in

septicemia, from Greek, meaning the state of putrefaction or decay. It can easily progress to a

deadly medical condition where the entire body is in an inflammatory state. Some cases of

infection can be gender-specific as well, because estrogen protects against the vibrio such as V.

vulnificus. Another major disease is cholera, an infection in the small intestine that results in the

inability to retain water. There is no antibiotic, and only limited success has been achieved in

creating a vaccine (MicrobeWiki, 2010).

A method of separating vibrio, cultivating them, and possibly even identifying them is

necessary in order to form generalizations and comparisons about them in different areas of the

Ala Wai Canal. This is possible through the selective medium Thiosulfate Citrate Bio Salts

Sucrose (better known as TCBS Agar). This can be bought premixed, or can be made concocting

per liter of deionized filtered water: 5.0g yeast extract, 5.0 pancreatic digest of casein, 5.0g digest

of casein, 5.0 peptic digest of animal tissue, 10.0g sodium citrate, 10.0g sodium thiosulfate, 5.0

ox bile extract, 3.0g sodium cholate, 20.0g sucrose, 10.0g sodium chloride, 1.0g ferric citrate,

14.0g agar, 40.0mg bromythol blue, and 40.0g thymol blue. The last two ingredients are

chemical and pH indicators respectively, and are the main reason why the end product is green in

color. This color shows that the pH of the finished medium is between 8.4 and 8.8 at room

temperature (25 degrees Celsius). This is apparent, because thymol blue turns yellow at very

acidic environments, and blue in very basic environments (BD, 2011).

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This medium specifically focuses and encourages vibrio growth because the bile salts

create an environment too high for most other bacteria to survive. Coupled with the perfect

temperatures and a rich organic environment causes vibrio also found in the Ala Wai to thrive.

Furthermore, seasonal shifts can also cause changes in the general amount of vibrio present in

water. This has to be taken into account and conclusions must not be immediately made after

only small samples are taken. TCBS agar ideally should be stored between 2 to 8 degrees Celsius

when not in use, away from direct light. In the best-case scenario, water samples should be

brought to the lab within three hours for investigation. A filter can be used to enumerate the

number of colonies that can be cultivated in this manner. The aerobic incubation period should

be at 35 degrees Celsius, typically between 18 to 48 hours.

TCBS agar is used to selectively isolate vibrio, for example, that causes cholera, food

poisoning, and diarrhea. The growth of V. alginolytics, V. cholerae, and V. paramaemolyticus, is

promoted by the medium, whereas bacteria such as Enterococcus faecalis and Escherichia coli

are inhibited. The agar was first concocted in 1963 by Kobayashi Nippon Saikingaku Zasshi

(Gomez-Gill, n.d).

In TCBS agar, V. chlolerae and V. alginolyticus turns yellow, whereas V.

parahaemolyticus and V. vulnificus remain green.

II. Materials & Method

TCBS Agar Plates, Incubator, Flask, Vortex Machine, Hockey Sticks, Micropipette

Take water samples from different locations in the Ala Wai Canal

Plate, dilute if necessary, and incubate for 24 hours at 37 degrees Celsius.

Note observations and growth on plates.

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III. Data

Sample Taken from Location(s): [A, Dock], [B, McCully Bridge]. Sample Set #1.

A 1/1 Dilution

8 Yellow Colonies, 8 Green Colonies

B 1/1 Dilution

7 Yellow Colonies, 9 Green ColoniesA 1/10 Dilution

0 Yellow Colonies, 1 Green Colony

B 1/10 Dilution

1 Yellow Colony, 1 Green ColonyNotes/Observations: 100 micro liters of sample spread per plate. Colony Size: Yellow ~0.5cm,

Green ~0.2-0.5cm each. Yellow colonies were seen to be, on average, bigger than the green

colonies. Halos were observed surrounding the yellow colonies.

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Data Set II

Sample Taken from Location(s): [3, Kaimuki], [|||3, Kaimuki], [4, Dock], [5, Top, Bridge].

Sample Set #2.

0 Yellow Colonies, 0 Green Colonies 0 Yellow Colonies, 0 Green Colonies

3 Yellow Colonies, 8 Green Colonies 7 Yellow Colonies, 10 Green ColoniesNotes/Observations: 100 micro liters spread per plate. Colony Size: larger than previous sample

set. Yellow colonies are about twice as large. Halos were observed again surrounding yellow

colonies.

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IV. Discussion

In the first sample set, water samples were taken from two areas of the Ala Wai Canal.

These areas are specifically the Dock and McCully Bridge. The samples of water were plated in

a 1/1 dilution as well as a 1/10 dilution. These samples showed that there was more growth in the

non-diluted samples rather than the diluted samples. There was about equal growth of bacteria at

the dock as well as the bridge. The samples showed 8 yellow and green colonies and 7 yellow

and 9 green colonies respectively. The 1/10 dilution plates had negligible growth with about 1

yellow and 1 green colony per plate.

The previous data led us to gather more data with only a 1/1 dilution. This time, it was

found that the area of the Ala Wai near Kaimuki had no bacterial growth. The bridge at the top as

well as the dock had significant growths however. The dock showed 3 yellow colonies and 8

green colonies and the bridge showed 7 yellow colonies and 10 green colonies. These two plates

showed bacteria about twice as big as before. The colony sizes are noted in the data portion of

the report.

Some notable things were that the water level on the second sample set was about a feet

lower. The tide was very low so that may have caused quite drastic differences. There was also a

halo that formed around the yellow colonies. Hyphae were also present in the samples in the

yellow colonies. The yellow colonies indicated V. alginolyticus or V. Cholerae.

Overall, there were almost always bacteria present in the Ala Wai, although they seem to

be scattered all over. Although there was not sufficient data, we found more bacterial growth

near the bridge at a lower tide. The colony sizes were about double the size as compared to the

first set of plates.

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V. Conclusion

Of all locations on the Ala Wai, the area near Kaimuki was the only place where there

was no growth in a 1/1 dilution.

According to our data, there are more pathogenic bacteria when farther away from the

dock. The dock plate only had a little growth.

When the tide was significantly lower (1 foot), our data showed that the bacteria were

twice as large.

There is not enough data to draw concrete evidence that these are the case. These

observations were made after two sets of samples were taken.

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Works Cited

BD (2006) TCBS Agar (for vibrio isolation). Retrieved [September 26, 2011], from Web site:

[http://www.bd.com/ds/productCenter/221872.asp]

CDC (2009) Vibrio Paramaemolyticus. Retrieved [September 26, 2011], from Web site:

[http://www.cdc.gov/nczved/divisions/dfbmd/diseases/vibriop/]

Gomez-Gil (n.d) Collection of Important Aquatic Microorganisms. Retrieved [September 26,

2011] from [CAIM] Web site: [http://www.ciad.mx/caim/TCBS.html]

Pmlmicro (n.d) Thiosulfate Citrate Bile Salt Sucrose (TCBS) Agar. Retrieved [September 26,

2011], from [Pmlmicro] Web site: [http://www.ehow.com/about_5435942_life-cycle-

vibrio-cholerae.html]

RD (2011) V. Alginoliticus. Retrieved [September 26, 2011], from Web site:

[http://www.rightdiagnosis.com/medical/v_alginolyticus_infection.htm]

Robinson (2011) The Life Cycle of a Vibrio Cholerae. Retieved [September 26, 2011], from

[eHow] Web site: [http://www.ehow.com/about_5435942_life-cycle-vibrio-

cholerae.html]

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