What are the uses & properties of a culture medium?

Microbiological culture media are used for the cultivation, isolation and identification of microorganisms – bacteria, yeast, molds, and fungi – derived from a variety of sources such as environmental samples, food, water and beverages, animal foods, cosmetic, pharmaceutical or clinical samples.

The culture media are designed to guarantee the viability of isolated microorganisms and are used for the enrichment of bacteria to acquire a sufficient amount of cells for further examination.

Microbiological culture media are defined according to ISO 11133-1 Guidelines and contain a large variety of ingredients to support the luxuriant cultivation of microorganisms:

    • Nutrients (e. g. Biological extracts, Digests of proteins)
    • Source of energy (e. g. Glucose)
    • Salts for maintaining adequate osmolarity (e.g. NaCl)
    • Enrichments (essential ingredients for auxotrophic or fastidious microorganisms)
    • Antioxidants and neutralizing agents against toxic substances like disinfectants (e. g. blood, coal, letheen)
    • Reducing agents to remove oxygen from media and maintain anaerobic or microaerophilic culture conditions
    • Indicator substances which indicate pH-shifts or cha nge of the redox state
    • Buffer for pH-regulation (e. g. potassium and sodium phosphates)
    • Chromogenic or fluorogenic nutrients, which are hydrolized by the microorganism and release a colored or flourescent hydrolysis product as indicator.
    • Selective agents to suppress the growth of unwanted microorganisms (e.g. chemicals, antibiotics, fungistatic agents)
    • Neutralizing agents for antibiotics or disinfectants (e.g. letheen, polysorbate) Solidifying agents (e.g. agar, gelatine) Water

Suitable production controls and sterilization conditions as well as a comprehensive quality control system are followed to assure reliable product stability.

How do I use the triple packed SCDA plates?

The triple packed Soyabean Digest Agar Plates are designed for use in Clean Rooms.

Micromaster offers irradiated triple packed media in 90 mm plates and as 55 mm contact plates for environmental monitoring and air sampling.

Properties of irradiated triple packed media plates:

Plates are packed in stacks of five and are labeled with product name, product code, expiration date and lot number for identification.

The filling volume of the plates reduces desiccation of the agar when used as settle plate or during active air sampling.

Plates are triple- packed with the inner bag containing a desiccant pouch, to minimize condensation.

Plates are gamma-irradiated in the final packaging at a dosage of 9-20 kGy to ensure the highest safety level.

The irradiated triple packed media plates may be stored at room temperature (20-25oC) and have a shelf life of up to 2-3 months.

The last inner bag is opened in the environmentally- controlled clean area to be monitored and the plates can be removed for use.

After removal of the plates, the inner bag with the desiccant should be removed from the area as soon as possible.

How do I store Micromaster culture media?

Storage of Dehydrated Culture Media:

The majority of Dehydrated Culture Media should be stored at room temperature within the range of 10-30°C in a dry place at a distance from sources of heat and sunlight; for a few, storage at 2- 8°C may be required.

Dehydrated culture media are hygroscopic. When bottles of dehydrated media have been opened for initial use, they should be tightly closed as soon as possible to protect them from hydration. The stock of dehydrated media should be rotated to ensure fresh product is in use.

The stocks should be maintained in a way so that the use of aged materials should be avoided, and the outdated media should be discarded.

The expiration date applies to the products in their intact containers when stored as directed. Do not use a product if it fails to meet specifications for identity and performance.

Prior to use, verify that the physical characteristics of the powder are typical. Hydration can lead to caking and/or microbial contamination which may render the culture medium unusable.

Storage of Ready-to-use Media:

Store Ready-to-use media at the indicated storage temperature and protect from light. Most media, and especially those containing dyes or indicators, should be protected from light during storage.

Store the media in a position, in which the label can be easily read.

Avoid storage temperatures below 2°C, because the agar in solid media might freeze. Even in 4°C-refrigerators there exist areas with lower temperatures. Frozen products should be discarded. Water is the main ingredient of culture media. To reduce condensation in the plates and the plastic bags, a desiccant has been added. However, fluctuations of temperature during transport and storage, which is the main source of condensation, should be avoided. The shelf life of the product is mentioned on the product label. Do not use expired culture media for microbiological examinations.

How do I prepare stock cultures and defined microbial suspensions for the QC of culture media?

Stock cultures and Passaging

American Type Culture Collection (ATCC®) or other reference culture collections provide test strains for growth promotion tests. Prior to preparing stock cultures, strain identity and homogeneity needs to be determined by the Quality Control Department.

At Micromaster : To prepare stock cultures, strains are resuspended in a suitable culture medium. This suspension is spread on a solid medium (e.g. Tryptic Soya Agar) to obtain single colonies. These are resuspended again in 20% sterile Glycerol broth. ‘N’ number of Seed Lot Cultures are created by aliquoting in 2ml amounts in sterile eppendorf vials / cryo vials and stored at temperatures below -20oC.

This procedure allows removing one vial at a time without thawing the stock culture (Passage-1). If desired, Master Cultures & Working cultures can be prepared & maintained for ease of use.

According to the Pharmacopeial procedure a test strain should be sub-cultured only a limited number of times before usage to obtain reliable quantitative results. Therefore a second culture (Passage -2) from aliquoted vials is prepared, from which vials can be removed continuously as per requirement to be cultured on solid media (sufficient for one year). From the cultures of Passage-3 or 4, fresh over night cultures are prepared in suitable broth before being employed in growth promotion testing.

A Schematic guide for use of reference cultures is given below. Specified concentrations of the test strains are required to determine growth promoting properties (< 100 cfu), growth inhibition (> 1000 cfu) and indicative properties (< 100 cfu) in most microbial enumeration tests, in tests for specified microorganisms, and in sterility testing. These defined suspensions are simply prepared by serial dilutions in sterile saline or sodium chloride peptone buffer according to the Pharmacopeias.

Preparation of spore suspensions of Aspergillus brasiliensis ATCC 16404

The culture from the second passage tube is streaked on Potato Dextrose Agar and incubated at room temperature for approx. one week to obtain strong sporulation (blackening of the colonies). A spore suspension is then prepared in Sterile Saline or Sodium Chloride Peptone buffer with 0.1% Tween 80, which can be stored at 4 -12°C for approx. 6 months. For growth promotion tests the spore suspension is diluted in Sterile Normal Saline or Sodium Chloride Peptone buffer to obtain 10-100 cfu in a defined volume.

Alternatively ready-to-use pellets containing a defined number of Aspergillus spores can be used to determine growth promoting or inhibitory properties of the culture media.

For most spore forming organisms like Bacillus subtilis ATCC 6633, Aspergillus brasiliensis ATCC16404, Clostridium Sporogenes ATCC 11437, etc. Spore suspensions can be prepared which can be stored up to six months.

Schematic Representation of Sub-culturing

What is the Method for handling and reconstitution of Dehydrated Culture Media?

The following considerations should be taken into account when sterilizing media that are autoclaved following the standard parameters of 121°C for 15 minutes:

The amount of heat applied during sterilisation is much greater than is necessary for destruction of the majority of organisms likely to be encountered. Tolerances of 15 minutes ± 1 minute combined with 121°C ± 2°C are considered appropriate.

Appropriate Quality Control checks should be carried out for temperatures which are outside 119 – 123°C, to ensure sterility and/or performance integrity.

Attention should be given to the media manufacturers’ instructions for use, in order to ensure optimal performance. Duration and temperature of sterilization are written on the Labels.

Certain media are more susceptible to over-heating, such as those with high sugar content, or those which contain inhibitory agents such as sodium desoxycholate or bile salts. The result of over-heating is a reduction in pH of the final medium.

Media containing carbohydrates should be autoclaved at a temperature not exceeding 116-118oC to avoid caramelization of the carbohydrate.

Do not autoclave media that should not be heat-sterilized. There are numerous formulations available that can merely be dissolved and used directly. The performance of such media is seriously impaired by subjecting them to heat.

The recommended sterilization times assume a volume of one liter (1000mL) or less. For larger volumes, the sterilization time should be extended but the temperature should not be raised. When larger volumes are used, validation studies should be performed to determine the optimum sterilization cycle for each unique container size/volume combination.

The sterilization temperature refers to the autoclave chamber not to the temperature of the medium. It is important that the time taken to reach the sterilizing temperature should be as short as possible.

Sterilization cycles must take into account suitable heat penetration times for example a one liter flask of medium should attain 121°C within 15 minutes of the chamber reaching that temperature.

The autoclave load and the relatively poor heat transferring properties of agar-containing media must be taken into account. The autoclave temperature probe is best located in the valve or drain, as this is the coolest part of the chamber and best represents the dynamics of the heating process.

It is important that physical parameters of the sterilizer and the efficacy of kill be monitored frequently through the use of calibrated instrumentation and biological indicators.

Can the selective supplements be stored after reconstitution?

Storage of reconstituted supplements is not a recommended procedure; however, at times there may be circumstances where some customers may wish to do this.

In such cases, aliquots of the freshly reconstituted supplement may be frozen at -20°C. When stored in this way the aliquots should remain stable for several months. When required, an aliquot should then be re-thawed only once and used immediately.

This procedure may require validation and it is recommended to do a performance check for the frozen aliquots against freshly reconstituted supplement.

Customers may thus produce their own guidelines for the shelf life of each supplement stored in this way.

What is the Shelf Life of Dehydrated Culture Media?

The expiry date mentioned on the label of Micromaster Dehydrated Culture Media refers to the shelf life of the product. The expiration date applies to the products in their intact containers when stored as directed. The total shelf life is typically 3-5 years.

Once opened, the total shelf life of Dehydrated Culture Media, Peptones and Hydrolysates is automatically reduced. The shelf life will depend on the frequency of opening of the container and the atmospheric conditions.

Deterioration of particularly hygroscopic products may be observed within six months of opening. Dehydrated media should be examined carefully after several months of opening to ensure maintenance of physical integrity and should only be used if they still appear free-flowing. Confirmation of the performance of selective and ‘shorter shelf life’ media, using positive and negative microbiological control strains, should be done after such extended periods of opening.

In order to maximise the shelf life of Dehydrated Culture Media, ensure that containers are not stored in areas of high humidity, e.g. autoclave areas.

What organisms do I need to use to QC the media?

Most of the Micromaster products have Technical Information Sheets which can be accessed on our website www.micromasterlab.com , or you may contact our Technical Team for the same. Each technical information sheet lists the organisms used at Micromaster for the Quality control of the medium.

Do I have to use all of the organisms listed on the Technical Information Sheet?

The end user is only required to check the performance of media using an organism that will produce a positive reaction and another organism that will produce a negative reaction for each reaction tested. Micromaster technical information sheets may include organisms to monitor weak positive reactions or at Micromaster we may test the growth of multiple organisms, but this testing may not be required to be done by the end user.

Is it alright to inoculate media plates right after taking them out of the refrigerator?

It is recommended to always warm the media to room temperature before inoculating. Some bacteria are sensitive to the cold (e.g., N. gonorrhoeae). Excess condensation on the surface of the plate can be evaporated by placing plates in the incubator for a short period.

Why should media be stored in the dark?

Some types of media contain dyes and other light sensitive ingredients. Excessive exposure to light can produce the formation of toxic peroxides that can inhibit growth. Hence it is recommended to store media away from the light, especially sunlight or UV light.