- 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.
- 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.
- Method of Use:
- Since the entire triple-packed product is subjected to a sterilizing dose of gamma irradiation, the contents inside the outer bag are considered to be sterile. This allows the inner bag to be aseptically removed and carried into an environmentally- controlled area without introducing contaminants.
- The outermost bag is removed in the First Air lock.
The second bag is removed in the next Air lock.
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.
- Storage of Dehydrated Culture Media:
- The majority of Dehydrated Culture Media should be stored at room temperature within the range of 10-30Â°C in a dry place at a distance from sources of heat and sunlight; for a few, storage at 2- 8Â°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 2Â°C, because the agar in solid media might freeze. Even in 4Â°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.
Stock cultures and Passaging
American Type Culture Collection (ATCCÂ®) 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. â€˜Nâ€™ 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 -12Â°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
Handling of Dehydrated Media Dehydrated media are hygroscopic and are sensitive to moisture, heat and light. They are adversely affected by the drastic changes in temperature i.e the hot/cold cycling temperatures which may occur between day and night laboratory temperatures.
The date of receipt of the container in the laboratory should be written on the label.
Storage conditions are usually indicated on the product label and should be followed. Store as directed on the label; usually below 25Â°C in a dry area, away from direct sunlight, autoclaves, drying ovens or other heat sources. Where indicated store at 2-8Â°C.
Check expiry date on the label, some media have significantly shorter shelf-lives than others.
Use stocks appropriately. Do not open a new bottle until the previous bottle has been emptied.Order the medium in appropriate pack sizes & quantities as per your requirements.
Note the date of opening of the container on the label. After use, make sure the container is tightly closed and return it to the designated storage area.
A medium in a large container which has been opened many times will deteriorate on storage.
Discard the medium if the powder is not free flowing, if the colour has changed or if it appears abnormal in any way Reconstitution of Dehydrated Media
Prior to use, examine the dehydrated material. Caked or discolored material should not be used for the preparation of culture media batches.
Complete instructions for the preparation of culture media are given on the label of each bottle.
As a general rule it is advisable to prepare one week’s requirement only.
Use water prepared by distillation, deionization or reverse osmosis. Check the pH of the water, if below 5.5, heat to drive off CO2 and re-check. The conductivity of the water should ideally be below 15 micro siemens. Rinse glassware before use.
Add the precise amount of powdered material to approximately one-half of the volume of purified water. After thorough mixing, add the remainder of the water with care being taken to wash down the sides of the container. This is an important step because dry culture media powder above the level of the water may not be sterilized in the autoclave and may be a source of contamination.
Preferably a flask / containers that are at least 2-3 times the volume of medium should be used to prepare media. Open the culture medium container away from moisture. Avoid inhaling the powder and prolonged
skin contact. Weigh the powder quickly, accurately and without creating clouds of dust’. Reclose the container as soon as possible.
Agar-free media will usually dissolve with gentle agitation.
Media containing agar should be heated to dissolve the agar before autoclaving. Bring the medium to the boil without scorching or burning. Exposure for longer periods can darken the medium and severely reduce its growth promotion properties.
Those media which should not be autoclaved will be ready to pour into sterile petri-plates or other sterile containers after this amount of heating.
Most culture media will require final sterilization in an autoclave at 121Â°C for 15 minutes.
The pH of the dehydrated medium has been adjusted so that the final pH of the prepared medium conforms with the label specification when the medium has been cooled to 25Â°C. Do not adjust the pH before sterilization.
The following considerations should be taken into account when sterilizing media that are autoclaved following the standard parameters of 121Â°C for 15 minutes:
The amount of heat applied during sterilisation is much greater than is necessaryÂ for destruction of the majority of organisms likely to be encountered. Tolerances ofÂ 15 minutes Â± 1 minute combined with 121Â°C Â± 2Â°C are considered appropriate.
Appropriate Quality Control checks should be carried out for temperatures which areÂ outside 119 – 123Â°C, to ensure sterility and/or performance integrity.
Attention should be given to the media manufacturersâ€™ 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,Â 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 (1000 mL) 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 sterilisation temperature refers to the autoclave chamber not to the temperature of the medium. It is important that the time taken to reach the sterilising temperature should be as short as possible.
Sterilisation cycles must take into account suitable heat penetration times for example a one liter flask of medium should attain 121Â°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.
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 -20Â°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.
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 â€˜shorter shelf lifeâ€™ 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.
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.
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.
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.
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.