TITAN MEDIA

Titan Medium or Dehydrated Culture Medium is a combination of complex nutrient substrates formulated for the cultivation of microorganisms. The components of a Dehydrated Culture Medium must satisfy the nutritional requirements of microorganisms, which in order to live and replicate require sources of nitrogen, carbon and trace elements. The trend has been towards using more defined media components with extracts and peptones taking the place of infusions and more recently, with chromogenic / fluorogenic compounds taking the place of carbohydrates and pH indicators.

1. PEPTONES AND EXTRACTS : 
   The peptones and extracts are nutrients obtained from meat, casein, soybean, yeast cells, liver, malt, gelatin, potato and groundnut etc. Peptones are produced by enzymatic hydrolysis of proteins. Enzymatic hydrolysis of proteins break the protein molecule at specific points and tends to preserve the amino acid and vitamin content of the original raw material while acidic hydrolysis breaks all the peptide bonds and produces free amino acids losing some important amino acids. The meat infusions and extracts obtained by protein coagulation through heating are very similar to peptones.


2. CARBOHYDRATES :
   Carbohydrates like glucose, lactose, mannitol and sucrose etc. are added to a Culture Medium as a source of energy to increase the rate of growth of microorganisms. They are present as fermentable substrates in combination with pH indicators for microbial differentiation.


3. INDICATOR SUBSTANCES :
   Indicator Substances can be classified into there types: pH indicators, oxidation-reduction indicators, and hydrogen sulphide indicators. Among the pH indicators, phenol red, neutral red, bromothymol blue and bromocresol purple have replaced the substances most used in the past such as litmus and Andrade’s indicator. The pH indicator reveals the formation of acids from carbohydrates and the formation of bases (Ammonium ions) from peptones, single amino acids or amines.
   
   Among the above-mentioned indicators, phenol red is the most sensitive since it reveals even very small variations in pH in the Dehydrated Culture Media and is most widely used.
   
   The oxidation-reduction indicators used in Culture Media are methylene blue and resazurin, which take on particular colourless to blue, and resazurin from colourless to pink.
   
   The hydrogen sulphide indicator substances are usually ferrous salts (ferric citrate, ferric sulphate, ferric ammonium sulphate, ferric ammonium citrate). The hydrogen sulphide produced by bacteria from sodium thiosulphate, reacts with the ferrous producing ferrous sulphide, which precipitates in the centre of the colony (characteristic colonies with black centres).


4. MINERALS :
   The salts (Mg, Mn, Fe, Ca, Zn, Cu) present in Dehydrated Culture Media provide necessary minerals for microbial growth. Potassium and sodium phosphates acts as buffering agents in the medium while sodium chloride maintains the osmolarity of the Culture Medium.


5. SELECTIVE AGENTS:
                 The selective agents are chosen and added to Culture Media to suppress the growth of unwanted organisms favouring the growth of desired ones. The first selective agents used in Microbiology were dyes and they are still present for this purpose in some formulations. Crystal violet is used in MacConkey agar to inhibit the Gram-positive bacteria, brilliant green is used in combination with bile salts to inhibit Gram-positive bacteria and stimulate the growth of Salmonella. Among the substances of biological origin, bile salts are the most widely used selective agents. They are present both in mixtures (Bile Salts, Bile Salt No. 3) and as pure substances (Sodium deoxycholate) to inhibit the growth of Gram-positive bacteria in media for isolation of intestinal microflora.
   
   A new class of selective agents, which is useful both in Clinical and Industrial Microbiology, are antibiotics. These substances, alone or in mixtures, already in the media or supplied separately as supplements, have the advantage of selecting the bacterial species to be isolated with greater specificity. Organic & inorganic salts make another class of selective agents. Sodium chloride at high concentration inhibits both the Gram negative and Gram-positive organisms, with the exception of Staphylococci Sodium azide in Dehydrated Culture Media is used for the selective isolation of Streptococci and Enterococci. Sodium Selenite in a buffered medium stimulates the growth of Salmonella and inhibits the Gram-positive bacteria. Sodium Citrate, Potassium tellurite, Sodium Tertrathionate and Sodium lauryl Sulphate also belong to this class of ingredients.


6. SOLIDIFYING AGENTS :
   The main solidifying agent in Dehydrated Culture Media is Agar. Robert Koch perfected the use of agar in culture substances and gave great impetus to the technique of isolation of microorganisms in pure culture. Agar is extracted from agarophyte seaweeds mainly Gellidium, Gracilaria, Plerocladia and Euchuma. Agar with different properties can be obtained based on the place of cultivation and techniques of extraction. The agar obtained from algae cultivated on the Altantic has a stronger solidifying character than agar obtained from the alage cultivated on the coasts of the Pacific. The agar in Culture Media has the unique role of a solidifying agent and has no nutritive properties as regard to microorganisms.
   
   Titan's Agar Quality is tested and certified under ideal conditions.
   
   Note: Repeated heating of agar medium decreases its Gel strength.


7. ENRICHMENTS :
   To improve the fertility properties of culture media for the cultivation of fastidious microorganisms (Neisseria, Haemophillus etc.) various enrichments are added normally after autoclaving and cooling the base medium to 50°C. Blood and animal serum are the most commonly used enrichments. According to the type of microbial research to be carried out heamoglobin, albumin, egg yolk, whole eggs, chemically defined enrichment solutions are used.


8. ENZYMATIC SUBSTANCE (CHROMOGENIC & FLUOROGENIC MEDIA) :
   Incorporating synthetic or natural substances, which can be cleaved in Dehydrated Culture Media by specific microbial enzymes, a big improvement has been obtained in the identification of some microbial species and genera. Depending on the substrate, cleavage of these products by microbial enzymes can lead either to a diffuse colouration or the formation of a coloured precipitate in the center of the colonies or, else to the formation of an opaque or clarification halo around the colonies for some media, identification occurs at the species level and for others at the genus or group level. The specificity of the detection is correlated to the specificity of the defected enzymatic activity and to the Culture Media formation. To increase the specificity and the sensitivity of the microbial detection in Chromogenic Culture Media, the enzymatic substrates are to be used in combination with optimized reagents, nutrient compounds and inhibitory substances.

DISSOLVING THE MEIDA
Weigh the required amount of Dehydrated Culture Medium and place it in an intact and clean container at least two three times larger than the final volume of medium to be produced. Use distilled or deionised water, that matches with the specifications of purified water of IP/USP/BP/BIS/EP

Add part of water and other permissible heat stable ingredients (if any) and swirl to dissolve. Add remaining water to make up the required volume. Gently heat the medium to boiling with frequent agitation to avoid overheating. Color may differ from other Brands but growth of microorganisms will be same up to desired level.

pH ADJUSTMENTS :
pH value of the culture media shall produce the equivalent value at 25°C as mentioned on the label. pH adjustment (if required) should be carried out with 0.1 N Hydrochloric Acid or Sodium Hydroxide Solution. For best results prepare the medium with distilled or deionised water only.

STERILIZATION :
Generally, sterilization is done at 15 psi (121°C) for 15 minutes using autoclave. Volumes larger than 2 liters may require more autoclaving time to achieve proper sterilization.

Pressure-Temperature Relations in Autoclave
(Figures based upon complete replacement of air by steam)

Pressure in Pounds	Temperature 0C 0F
5 10 15 20 25 30	108 116 121 127 131 134	226 240 250 260 267 274

DISPENSING OF THE MEDIA:
Cool the agar based medium to 50°C and aseptically add heat labile or heat sensitive enrichments or supplements sterilized separately by suitable methods. For slants & motility media prepare in tube and keep the tubes in slanted position. Sterile broths may be cooled to room temperature before addition of the supplements.

Dispense the medium into the sterile petridishes, tubes etc. as per the requirements and immediately plug or recover.

STORAGE OF PREPARED MEDIA:
If the prepared media are not to be used within 24 hours then these should be stored at low temperature (2-8)°C in moisture proof containers. Petriplates should be inverted and the caps of the tubes be tighten firmly before storage.

DISPOSAL OF MEDIA:
All samples and cultures should be handled carefully and should not be disposed without autoclaving. These should be discarded after autoclaving at 15psi (121° C) for 15 minutes.

STORAGE OF MEDIA:
The Dehydrated Culture Media needs to be stored below 25°C to maintain shelf life of products.

CERTIFICATE OF ANALYSIS:
Certificate of Analysis (COA) of every lot released for market is issued by the company, can be sent to customers on request.

TSE/BSE CERTIFICATE: Certification for TSE/BSE issued by the company on request.