RESULT

Figure 1 Microgen GN-ID A+B Report Form.

Figure 2 Results for sample organisms cultured in trypticase soy broth (TSB)
containing 0%, 2%, 6%, 8% and 10% NaCl respectively.
DISCUSSION
The organism determined using the Microgen Identification System Software is V.
parahaemolyticus with a probability of 84%. It is an oxidase positive and motile
organism. It also showed positive result for the nitrate reduction test. The other result for
all the reactions in the microwell plates were as observed and recorded in Figure 1.

The sample of V. parahaemolyticus were cultured in trypticase soy broth (TSB)

containing 0%, 2%, 6%, 8% and 10% NaCl respectively which were incubated at 37C
for 2 days. However, TSB containing 2% NaCl was incubated separately at 42C
temperature. As seen in Figure 2, the result shows that only the tubes of TSB containing
0% and 6% NaCl have a positive reaction and appeared to be turbid. According to Paydar
2013, V. parahaemolyticus are able to multiply very rapidly in a medium cantaining 3 to
5% NaCl and they can tolerate up to 8% NaCl but are sensitive to 10% NaCl. Hence, this
correspond to the result obtained in this experiment as there are most growth observed in
6% NaCl and no growth is recorded in 10% NaCl. There is no growth in 8% NaCl as well
due to its unfavourable condition. For the tubes with 2% NaCl, there is no growth
observed as well because V. parahaemolyticus is only able to grow in temperature range
of 5-42C (Paydar 2013). Hence, the tube which is incubated at 42C is also an
unfavourable growth condition for the cell.

The isolation procedures for V. parahaemolyticus can be performed using

conventional cultural methods (Elliot et al. 2001), optimally adapted to water samples.
Seawater is first filtered through pore size polycarbonate membranes and then incubated
in alkaline peptone water at 36 1C. After 24 hours of incubation, a loopful of
enrichment broth is streaked onto thiosulfatecitratebilesucrose (TCBS) agar and then
incubated at 37C for another 24 hours. Phenotypic identifications are then performed
using the following three steps: to confirm the typical traits of the Vibrio genus
(screening phase), the strains cultured on 3% NaCl TSA (36C) are subjected to a set of
six tests (Gram staining, oxidase test, fermentative degradation of dextrose, nitrate
reduction, motility test and growth under anaerobic conditions) (Elliot et al. 2001). In the
second phase, bacterial strains confirmed as Vibrio were subjected to the salt tolerance
test in 0%, 6%, 8% and 10% NaCl tryptone water, as well as 2% NaCl growth at 42C.
Finally, the strains presumptively identified as V. parahaemolyticus are subjected to
biochemical identification using commercially available miniaturized systems API 20E.

V. parahaemolyticus is a ubiquitous human pathogen which may cause

gastroenteritis when contaminated raw, insufficiently cooked or postheat-contaminated
seafoods are consumed (Paydar 2013). It is a major food-borne pathogen that causes
worldwide health problems (Hara-Kudo 2001). Since this species is highly abundant in
marine products, they have become a major concern in the production and trade of
seafood. Established food safety measures are required to be conducted for marine
products, as the main source of a large number of pathogenic bacteria, including V.
parahaemolyticus.

The correlation has been well established that V. parahaemolyticus strains that
cause illness in humans is almost always Kanagawa-positive and isolates recovered from
seafood are almost always Kanagawa-negative (Kaysner and DePaola 2004). Kanagawa
reaction is the hemolytic phenomenon in which a thermostable extracellular substance is
responsible for the hemolytic reaction. The hemolysin responsible for the Kanagawa
reaction is a thermostable direct hemolysin (TDH). It plays a role in the pathogenesis of
the vibrio gastroenteritis.

REFERENCES

Elliot, E.L., Kaysner, A.C., Jackson, L. and Tamplin, M.L., 2001. Vibrio cholerae, V.
parahaemolyticus, V. vulnificus, and other Vibrio spp. USFDA bacteriological
analytical manual, 8th ed. Gaithersburg: AOAC International, 901-927.

Hara-Kudo, Y., Nishina, T., Nakagawa, H., Konuma, H., Hasegawa, J. and Kumagai, S.,
2001. Improved method for detection of Vibrio parahaemolyticus in seafood.
Applied and enviromental microbiology, 67(12), 58195823.
Kaysner, C. and DePaola, A., 2004. Bacteriological analytical manual: Vibrio [Online].
Available from: http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods
/ucm070830.htm [Accessed: 6 Oct 2015].

Paydar, M., 2013. Isolation and differentiation of Vibrio species from seafood and
molecular characterization of Vibrio parahaemolyticus. Dissertation (M.Sc).
University of Malaya.