culture media products are made of high quality ingredients so as to produce maximum good results as expected by our customers. It is of great significance to know the functions and reactions of a particular ingredient and its interaction with other ingredients in a media. Here arises the necessity of strict evaluation of the ingredients.
In each and every ingredient undergoes thorough testing prior to manufacturing by our well-equiped and highly efficient quality control department. Maximum quality control measures are taken throughout the manufacturing process to give the desired results. It will be our greatest effort to provide products of maximum attainable quality to our customers.
We serve a wide range of culture media products to meet the var ious requirements of Dairy , Pharma ceuticals , Veterinary, Cosmetics, Water Treatment, Public Health and Medical Microbiology departments.
These were originally supplied in the form of meat infusion, Beef or yeast extracts frequently replace meat infusion in culture media. The addition of peptones, which are digests of proteins, provides readily available sources of nitrogen and carbon.
The pH of the culture medium is important for microorganism growth. Temperature is another important parameter mesophilic bacteria and fungi have optimal growth at temperatures of 25-40 C; thermophilic bacteria ("heat loving") grow only at temperature greater than 45 C psychrophilic ("cold loving") organisms require temperatures below 20 C. Human pathogenic organisms are generally mesophiles.
COMMON MEDIA CONSTITUENTS
Media formulations are developed on the ability of bacteria to use media components.
|Amino-Nitrogen||Peptone, protein hydrolysate, infusions and extracts|
|Growth Factors||Blood, serum, yeast extract or vitamins, NAD|
|Energy Sources||Sugar, alcohols and carbohydrates|
|Buffer Salts||Phosphates, acetates and citrates|
|Mineral Salts and Metals||Phosphate, sulfate, magnesium, calcium, iron|
|Selective Agents||Chemicals, antimicrobials and dyes|
|Indicator Dyes||Phenol red, neutral red|
|Gelling agents||Agar, gelatin, alginate, silica gel|
Peptone, protein hydrolysates, Infusions and extracts are the major sources of nitrogen and vitamins in culture media. Peptones are water-soluble ingredients derived from proteins by hydrolysis or digestion of the source material, e.g. meat, milk.
Carbohydrates are employed in culture media as energy sources and may be used for differentiating genera and idenifying species.
Buffers maintain the pH of culture media.
Selective Agents include Bile Salts, dyes and antimicrobial agents.
Bile Salts and desoxycholate are selective for the isolation of gram-negative microorganisms, inhibiting gram-positive cocci.
Dyes and indicators are essential in the preparation of differential and selective culture media. In these formulations, dyes act as bacteriostatic agents, inhibitors of growth or indicators of changes in acidity or alkalinity of the substrate.
Antimicrobial agents are used in media to inhibit the growth of bacteria, yeasts and fungi.
Solidifying agents, including agar, gelatin and albumin, can be added to a liquid medium in order to change the consistency to a solid or semisolid state.ENVIRONMENTAL FACTORS IN CULTURE MEDIA
Many microorganisms require an environment of 5-10% CO2. Levels greater than 10% are often inhibitory due to a decrease in pH as carbonic acid forms. Culture media vary in their susceptibility to form toxic oxidation products if exposed to light and air.
Proper moisture conditions are necessary for continued luxuriant growth of microorganisms. Organisms require an aqueous environment and must have "free" water. "Free" water is not bound in complex structure and is necessary for transfer of nutrients and toxic waste products. Evaporation during incubation or storage results in loss of "free" water and reduction of colony size or total inhibition of organism growth.
Protective Agents and Growth Factors
Calcium carbonate, soluble starch and charcoal are examples of protective agents used in culture media to neutralize and absorb toxic metabolites produced by bacterial growth.
NAD (V factor) and hemin (X factor) are growth factors required by certain bacteria; e.g., Haemophilus species and for enhanced growth of Neisseria species.
Surfactants, including Polysorbate 80, lower the interfacial tension around bacteria suspended in the medium. This activity permits more rapid entry of desired compounds into the bacterial cell and can increase bacterial growth.
The preparation of culture media from dehydrated media requires accuracy and attention to preparation. The following points are included to aid the user in successful and reproducible preparation of culture media.DEHYDRATED MEDIA AND INGREDIENTS
BACTERIA CONTROL STRAIN SOURCE
An integral part of quality control testing include quality control organisms. Microorganisms should be obtained from reputable sources, for example, the American Type Culture Collection (ATCC ) or other commercial sources.
MAINTENANCE / FROZEN STOCK CULTURES
If using commercial stock cultures, follow the manufacturer's recommendations for growth and maintenance.
To prepare frozen stock cultures of Staphylococcus species, Streptococcus species, Enterobacteriaceae and Pseudomonas aeruginosa :
TO USE A FROZEN CULUTRE :
Prepare no more than three serial subcultures from a frozen stock culture.
Maintain anaerobic cultures in Cooked Meat Medium or another suitable anaerobic medium. Alternatively, use frozen anaerobic cultures.TEST PROCEDURE
TO TEST CULTURAL RESPONSE
Dilute the cell suspension 1:100 in normal saline or purified water. Inoculate each plate with 0.01 ml to give 1-2 x 10 CFU/plate. Reduce the inoculum ten fold, if necessary, to obtain isolated colonies.
SELECTIVE MEDIA AND TUBED MEDIA
Dilute the cell suspension 1:10 in normal saline or purified water. Streak each plate with 10.01 ml of the suspension to provide 1-2 x 10 CFU/plate. Reduce the inoculum ten fold, if necessary, to avoid overwhelming some selective media.
For general-purpose media, sufficient, characteristic growth and typical colony morphology should be obtained with all test strains. For selective media, growth of designated organisms is inhibited and adequate growth of desired organisms is obtained. Color and hemolytic reaction criteria must be met.
National Committee for Clinical Laboratory Standards. 1996. Quality assurance for commercially prepared microbiological culture media, 2nd ed. Approved standard. M22-A-2, vol. 16, no. 16. National Committee for Clinical Laboratory Standards, Wayne, PA.
"Typical" chemical compositions have been determined on media ingredients. The typical analysis is used to select products for research or production needs when specific nutritional characteristics are required. The specifications for the typical analysis include :
All values are presented as weight/weight; % = g/100 g.
The higher the ash content, the lower the clarity of the prepared ingredient. The ash content includes sodium chloride, sulfate, phosphates, silicates and metal oxides. Acid-insoluble ash is typically from silicates found in animal fodder.
Lower moisture levels (<.5%) are preferred. Higher moisture levels in dehydrated ingredients may reduce stability. In the presence of high moisture and high ambient temperatures, chemical interactions will cause darkening of the product and falling pH. These characteristics indicate product deterioration.
Total Nitrogen : Total nitrogen is usually measured by the kjeldhal digestion or titration method. Not all organic nitrogen is nutritive. Percent (%) nitrogen x 6.25 ~ % proteins, peptides or amino acids present.
Amino Nitrogen : The amino nitrogen value shows the extent of protein hydrolysis by measuring the increase in free amino groups. This is a nutritionally meaningful value.
Changes in pH from specified values, either after storage or processing, indicate deterioration. These changes are uaually accompanied by darkening of the end product. Hydrolysates vary in their pH resistance according to their inherent buffering (phosphate) capacity.
High-phosphate ingredients may be unsuitable for pH indicator media due to the inherent buffering of phosphates.
However, phosphates do aid in gas production, which can be enhanced by deliberate addition of sodium phosphate.
The NaCl content may reflect significant pH adjustments during processing e.g., acid hydrolysates. (See Ash).
Trace metals can directly antagonize antimicrobial activity in vitro or impact toxin production, chelating agents may be added to culture media to sequester trace metals and clarify the media.
The Dehydrated Media are highly hygroscopic and get deteriorated easily unless stored in a cool & dry place away from bright light.
So care must be taken in the storage of dehydrated media.
Following instructions should be followed while using dehydrated culture media.
1. Read carefully the instruction given on the label.
2. Note the batch no. and date before opening the container.
3. Before use confirm that the media is not deteriorated physically.
4. Since the media is hygroscopic in nature, please ensure that the container is tightly closed and stored in cool and dry place after use.
DISSOLVING THE MEDIA : For dissolution use clean undamaged glassware two-three times larger in volume than the final volume of media to be prepared. Water which meets the
U.S.P./I.P specifications should only be used.
Place accurately weighed amount of the medium in a clean dry flask. Add part of water and swirl until dissolved then add the remaining water through the sides of the flask to make
up the required volume. For complete dissolution heat the media taking care to avoid overheating or scorching.
ADJUSTMENT OF pH : After preparations the pH of the media should be in concordance with value mentioned on the label at 25°C. If required, the pH should be adjusted to the
specified value by adding 1N or 0.1N HCI or NaOH solution.
STERILIZATION : Sterilization of the media is usually carried out at 121 °C for 15 minutes using an autoclave. Autoclave efficiency should be checked from time to time.
POURING OF STERILIZED MEDIA : After sterilization Agar media should be poured into petri dishes at 45-50°C. The medium should be mixed well avoiding bubble formation.
Agar surface should be dried at 30-40°C in the incubator before inoculation to avoid microbial swarming.
STORAGE OF PREPARED MEDIA : Unless the medium is not used in the same day it is prepared, then it should be kept in moisture proof containers.
Agar containing media should not be stored at higher temperatures. Agar plates should be stored at 2-8°C in sealed moisture proof containers. Stability of the prepared culture
media is limited and varies considerably. Never store media below 0°C as it destroys its gel strength.
DISPOSAL OF MEDIA : All specimens and cultures should be carefully handled and must not disposed without autoclaving. Cultures in vessels should be autoclaved for
approximately 30 minutes at 121 °C before disposal.
STORAGE OF MICROGEN CULTURE MEDIA PRODUCTS : For obtaining desired results style="margin: 0px 6px" src="images/micrigen-logo1.png"> culture media products should be stored in specified storing conditions. It is
recommended to use the products in the order of Batch number/Mfg. date.
Storage temperature and shelf-life of the style="margin: 0px 6px" src="images/micrigen-logo1.png"> culture media products are as follows.
(a) Dehydrated media : Dehydrated media if stored under specified conditions will have shelf-life of 2 to 5 years. Storage temperature for dehydrated media are preferably
between 8 and 25°C.
(b) Selective supplements : Except Horse serum (-20°C) all other supplements are required to store at 2 - 8°C. These products have a shelf-life of 1 - 3 years.
(c) Antimicrobial susceptibility discs : These are to be stored at -20°C but working stock at 28°C Shelf-life is from 1 to 3 years.
certain precautions while using dehydrated media
|Drift in pH hydrolysis of||Overheating, incomplete mixing, prolonged sterilization, use of alkaline glass, impure water, ingredients, prolonged storage at high temperature.|
|Incomplete Solubility||Inadequate heating of agar media incomplete mixing.|
|Darkening||Overheating of the medium, excess amount of dehydrated powder, improper mixing.|
|Soft Gel||Agar not in solution, improper reconstitution of dehydrated medium, acid hydrolysis of agar, failure
to compensate for dilution of agar by the incoclum.
|Loss of Growth differentiating properties||Promoting of repeated remelting, excessive heating, incomplete mixing, failure to compensate for dilution of ingredients, disturbance in the formula by inoculum carriers, etc.|
|Abnormal Colour of Medium||Deteriorated, dehydrated medium, improperly washed glassware, impure water.|
|Contamination||Improper/Insufficient sterilization. Poor technique in adding enrichments and pouring plates.|