Saburo OHKAWA, Director of Ohkawa Microorganism & Medium Laboratory
There are many types of media for the detection of Vibrio parahaemolyticus; its selective enrichment media are alkaline peptone water medium, Monsur medium, etc., its selective isolation media are TCBS agar, Vibrio agar, Taurocholate Tellurite Gelatin Agar（TTGA）, VP agar, VPSA agar, CHROMagar, X-VP agar, ES Vibrio agar, and those for a bacteria count system are DOX medium for vibrio and DOX medium for Vibrio parahaemolyticus.
In Japan, culture media with enzyme substrate such as TCBS agar and CHROMagar are used as selective media for Vibrio parahaemolyticus. I will deal with TCBS agar in this piece of column.
TCBS agar（Thiosulfate citrate bile salts sucrose agar）
TCBS agar, developed by Kobayashi, Enomoto, Sakazaki, Kuwahara, et al., is a selective isolation medium for vibrio including Vibrio cholera and Vibrio parahaemolyticus. This medium is characterized by sodium chloride concentration and high pH level.
Vibrio, halophilic bacteria, need sodium chloride to grow (they cannot grow in media without sodium chloride). Therefore, TCBS agar contains 1.0g/L of sodium chloride.
The pH level at which vibrio can grow is 5.6-9.6 and its optimum is 8.0. On the other hand, the optimum pH for many other bacteria falls within 6.8-7.6 and their growth will be inhibited at pH outside this range. As this medium is pH 8.6, bacteria other than vibrio cannot grow.
If sodium cholate (bile salt), a selective agent, coexists with sodium citrate, it inhibits growth of many types of bacteria (including vibrio) at pH 7.0. At pH 8.6, however, many types of bacteria are inhibited to grow but vibrio is an exception.
Also, fermenting and non-fermenting bacteria of sucrose can be distinguished according to the color of colonies. Vibrio parahaemolyticus (without sucrose-fermenting function) forms green colonies and Vibrio cholera (with sucrose-fermenting function) forms yellow ones.
2. Approximate formula for per Liter
|Pancreatic digest of casein||5g|
|Papaic digest of animal tissue||5g|
3. Functions of the medium components
Pancreatic digest of casein, papaic digest of animal tissue
Essential nutrients for bacterial growth are ①nitrogen source and ②carbon source. Bacteria cannot break down protein unless it is digested or decomposed to be the form of polypeptide or peptide. A substance generated from digestion or decomposition of protein is called peptone. There are several types of peptones such as casein peptone, soy peptone, meat peptone, myocardial peptone, and gelatin peptone. In TCBS agar, casein peptone (pancreatin digestion of the pancreas) and papain digestion of animal tissue are used. Casein peptone is used as a basal peptone due to its economical superiority. Papain digestion of animal tissue acts as a growth-promoting substance like an extract because it is rich in carbohydrate, vitamin and mineral.
Yeast extract is not an essential nutritional element for general bacteria to grow. It is usually used as a supplementary nutrition of peptone. Adding extracts will make up for the loss of nutrients and promote bacterial growth. Also, vitamins in extracts are rich in coenzymatic function (increasing enzymatic activity of bacteria).
Sucrose is added for the purpose of ①releasing energy from carbon source and ②distinguishing bacteria through decomposition of carbohydrate. This enables TCBS agar to indicate whether a bacterium has a sucrose fermentation function or not. Vibrio parahaemolyticus does not ferment sucrose, whereas Vibrio cholera does. Thus these two types of bacteria can be distinguished.
Bovine bile, sodium cholate
The purpose of adding these substances is ①inhibiting the growth of Gram-positive bacteria and yeast-like fungus (Gram-positive bacteria are lysed by surfactant and cannot grow because they have thick peptidoglycan layer and no extracellular membrane, whereas Gram-negative bacteria have thin peptidoglycan layer and thick extracellular membrane which prevents the lysis and growth-inhabitation), and ②inhibiting the swarming of proteus.
Bile salts used in culture media are sodium deoxycholate, sodium cholate and sodium taurocholate. Although sodium deoxycholate has the best selectivity among these bile salts, it is unstable in media and easy to crystallize by too much heating or sudden temperature change. Therefore, mixed bile salts of sodium deoxycholate and cholic acid in a proportion of 6:4 are generally used.
Ferric citrate, sodium citrate
These are used as pH adjuster (citric acid, trisodium citrate, etc.)
Ammonium ferric citrate neutralizes and detoxifies deposited bile acid. Sodium citrate increases solubility of bile acid (sodium cholate) in culture media and inhibits the growth of Gram-positive bacteria.
Sodium thiosulfate is used as a sulfur source. Also, coexisting with sodium citrate, bile, and cholic acid, it inhibits the growth of Gram-positive bacteria and coliforms when medium is alkaline.
Brom thymol blue (BTB)
BTB, a type of pH indicator, is a pigment whose color changes depending on pH. As sucrose-fermenting vibrio grown in this medium produce lactic acid, BTB will turn yellow. Bacteria that do not ferment sucrose will form green and transparent colonies due to alkalinization by degradation of peptone in the medium. BTB can take both protonated and deprotonated states, which changes its color tone. It turns yellow at pH < 6.0, blue at pH > 7.6, and green in between. Also, it turns red with very strong acid, and purple with very strong base.
Thymol blue (TB)
TB, a type of pH indicator, is a pigment whose color changes depending on pH. Its color changes in two stages. It turns red at pH 1.2 or below and yellow at pH 2.8-8.0. Also, it turns blue at pH 9.6 or above.
Agar is used to solidify media. Raw material of agar is seaweed such as Gelidiaceae and Gracilaria vermiculophylla. The latter one is generally used to make agar for a culture medium (for its reasonable price). A principal component of agar is agarose with linear sugar, which makes bacterial degradation difficult. Agar can contain relatively high weight of water molecules and has a spongy structure. It can maintain water and store nutrition in it. For that reason, agar is suitable for a culture medium of bacteria. The temperature at which agar medium begins to melt by heating is called “melting point”, and that melted agar medium begins to solidify is called “congealing point”. The melting point of agar is 85-93 °C and the congealing point is 33-45 °C. These figures vary according to the ingredients mixed into agar. Quality of agar determines quality of the medium. Fine agar has superior transparency, jelly strength, viscosity, and water retentivity.
4. How to use
① Culture test fluid in selective enrichment media (alkaline peptone water) for 18-20 hours at 37°C.
② Collect 1 loopful of culture liquid and streak-smear it on TCBS agar.
③ Culture it for 20±2 hours in an incubator of 35±2°C.
④ Determine whether or not colonies are present.
As for grown colonies, conduct a textbook-based test for identification of vibrio.
5. <Quantitative culture> # Conducted if positive in a qualitative method.
⑤ Dilute 10% food emulsion tenfold in phases.
⑥ Deliver 0.1ml of each diluting phase by drops onto Thiosulfate citrate bile salts sucrose agar and spread it with a bacteria spreader.
⑦ Culture them for 48hours at 37°C.
⑧ Count colonies and figure out the bacterial count per gram.
＊Needs to be identified.
6. Limitation of this culture medium
1. Bacteria other than vibrio, such as K.pneumoniae, Proteus spp., Pseudomonas spp., Aeromonas spp., Enterococcus spp., and Candida spp., may grow in this medium. Especially K.pneumoniae forms yellow colonies whose size is remarkably similar to those of Vibrio cholera. This tendency becomes more pronounced as days go by after producing the medium. Grown colonies need to be identified.
2. Slow-growing Vibrio cholera or those with weak sucrose-fermenting function may form green or colorless colonies in TCBS agar. Identification of colonies requires attention.
3. Although many types of vibrio grow in this agar, some types with high nutritional requirement, such as V.hollisae and V.metchnikovii, do not. TCBS agar is not suitable for the detection of these bacteria.
4. Pathogenic vibrio (such as Vibrio parahaemolyticus and Vibrio cholera) are present in small numbers in a test sample. They cannot be detected in some cases due to overgrowth of V.alginolticus. This bacterium grows actively and has a strong sucrose-fermenting function. It changes the whole medium yellow and Vibrio parahaemolyticus, small in number, cannot be detected.
5. Grown colonies in TCBS agar, which were yellow right after being taken out of an incubator, may turn green when left at room temperature (ex. Vibrio cholera).
6. An oxidase test of grown colonies results inaccurately. Especially sucrose-fermenting bacteria (yellow colonies) show false-negative reaction.
7. Grown colonies cannot be used for an agglutination test with diagnostic immune serum (Vibrio parahaemolyticus, Vibrio cholera, etc.) ⇒ shows no or weak agglutination.
8. Inaccurate measurement of vibrio count may occur. Damaged vibrio in food grow insufficiently. If cell membrane or cell wall of a bacterium in food is damaged by heating, drying, freezing or other production processes, the bacterium will be susceptible to sodium cholate and bovine bile in this medium and its growth will be insufficient. Also, this medium is not suitable for the detection of Vibrio parahaemolyticus in frozen fish and shellfish. Pre-culture is needed using an appropriate liquid medium.
9. Performance of TCBS agar differs greatly according to production company and lot. Quality control of the medium is required when using it. Ability for growth support and selectivity, above all, of this medium will decrease significantly with passing of time, storage condition, etc.
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