Vibrio Cholerae - Determination Of Vibrio Cholerae in the Milk Sample

Microbiologi Report

Purpose                       : To determination number of Vibrio Cholerae bacterium in the milk  sample                                                

Principle                     :
Growth of Vibrio cholerae is characterized by the formation of yellow colonies on media TCBSA obtained and incorporated into the NA media, incubated at 37 ℃ for 24v hours. Suspense was transferred to the TSIA and VP of media were incubated at 37 ℃ for 24 hours.

  

Theorical Basic :

Vibrio Cholerae

Vibrio cholerae is a gram-negative bacteria, shaped bacilli (rod) and are motile (can move), has the structure of the antigen flagelar antogenik H and O somatic antigen, gamma-proteobacteria, mesophilic and kemoorganotrof, berhabitat naturally in aquatic environments and is commonly associated with eukaryotic . The species is frequently associated with Vibrio patogenisitasnya properties in humans, especially V. cholerae causes cholera disease in developing countries which have limitations for clean water and have poor sanitation.
Scientific classification :
Kingdom          : Bacteria
Phylum            : Proteobacteria
Class                : Gamma Proteobacteria
Order              : Vibrionales
Family             : Vibrionaceae
Genus              : Vibrio
Species            : V.cholerae

These bacteria can grow in media with high pH (9 to 9.5); can ferment glucose, lactose & fructose without the aid of acid sulfide; grown on media containing thiosulfate, citric & gall; active motility; positive oxidase reaction on the test; grams negative; curved rod-shaped; has several types based on antigenic differences in the O-antigen, three of this type is the Inaba, Ogawa & hikojima. Power is transmitted bacterial pathogen as dirt mouth disease and acquired by ingestion of contaminated food & water impurities. These organisms are not spread beyond the gastrointestinal tract, where the organisms have multiplied to a very high concentration in the small intestine and colon. Vibrio cholerae remain firmly attached to the intestinal mucus layer.

To perform the isolation and maintenance of vibrio, can use the media Thiosulfate-citrate-bile salts agar (TCBS) which is a selective medium for the isolation and purification of Vibrio. Vibrio able to use sucrose as a carbon source is yellow, while the other is green.

However, there are several microbial that can also grow on this medium, such as Staphylococcus, Flavobacterium, Pseudoalteromonas, andShewanella. [2] As for the multiplication of Vibrio, the media can use Alkaline Peptone Water (APW), which has a relatively high pH, ​​which ranges 8.4 and containing 1-2% NaCl. [2] The vibrio is the optimum growth at temperatures ranging between 20 - 35^oC.

Techniques used in the identification of phenotypes V. cholerae is a test of lysine and ornithine decarboxylase (arginine) decarboxylase, oxidase, Kliger Iron Agar (KIA), and indole test. V. cholerae will show positive results on these four biochemical tests. The results of testing positive for oxidase and lysine and arginine decarboxylase test is the formation of dark purple color. At the MCH test, no gas is formed, with a slant (the surface of the media), color red (alkaline) and butt (bottom medium) yellow (acidic). For the indole test, purplish-red color is formed on the surface. Vibrio cholerae (V. cholerae) is a gram-negative, short rod-shaped or comma, can ferment sucrose on TCBS media and be able to move because of the polar flagella.

Media

Media is a collection of organic substances that are used to grow bacteria with certain conditions, therefore the culture media should contain enough nutrients for bacterial growth (Jackson, 2000). In addition to temperature and pH should be appropriate (Karsinah et al, 1994) is also worth noting about the pressure of osmose and sterility (Anonymous, 2000).
Media is distinguished over the shape, structure, and properties of the media:
a. According to the known shape of :
1. Solid media, if media is added in between 12-15 grams of agar-agar powder / 1000 ml of media.
2. Liquid media, if the substance is not added into the media junkie.
3. Semisolid or semi-liquid, if the addition of compactor only 50% or less than it should.
b. According to its structure:
1. Natural media, the media are prepared by natural ingredients.
2. Synthesis media, ie media prepared by chemical compounds.
3. Semi-synthetic media, the media are composed of natural materials and semi-synthetic ingredients.
c. According to its nature:
1. General media, the media can be used for growth and development of one or more groups of microbes.
2. Rich media, to get the growth of certain types of bacteria that do not grow on simple media.
3. Selective media, ie media covered only zero or one particular type of microbe, but will inhibit or lethal to other strains that are not expected. Media eg MSA (Mannitol Salt order).

Media TCBSA

Erlenmeyer containing 300 ml of distilled water, sterilized in an autoclave, then didindingkan. TCBSA media weighed as much as 26.4 grams put into Erlenmeyer that has been sterilized. Then the Erlenmeyer containing media reheated over a water bath to dissolve all. Then the media is poured into petridisk then cooled.
 

Tools and Materials :

Ø Tools :

Test tube
Petrididk
600 ml beaker
Pipette 10 ml scale
300 ml Erlenmeyer
Enkas
Ose
Incendiary spritus
Laminar flow of water

Ø Material :

Media TCBSA
Media NA
Media TSIA

Procedures :

-          10 ml sample pipetted into Erlenmeyer syrup containing diluent solution APW, Kemudiuan incubated for 1-2 x 24 hours at 37 ℃.

-          Then a solution of APW ± 1ml pipetted into the medium and then scratched TCBSA
After that TCBSA media that already contains the sample incubated for 1-2x24 hours at 37 ℃

-          Then the formed colonies were observed when yellow, it means that the positive
Of culture on media TCBSA the scratched on NA media, incubated 1-2 x 24-hour diving [at 37 ℃

-          Of culture on NA media taken 1 sengkelir OSE and the scratched on sloping TSIA aga and VP inkubasikan an x24-hour immersion at 37 ℃

-          After incubation time, into the VP was added 0.6 ml laruta \ n alpha naphtol and 0.2 ml of aqueous KOH

Observation : 

In TCBSA media were incubated for 1 x 24 hours at a temperature of 370c formed yellow colonies (+).
In biochemical tests were incubated 24 hours at a temperature dive 370c in which the media TSIA and VP positive.

Conclusion:

From the above observations it can be concluded that the samples of milk powder on testing Vibrio Cholera, yellow colonies formed TCBSA media and on biochemical tests TSIA and VP positive.