English: Cultivation of microorganisms: nutrient media

- Preparation of solid and liquid nutrient media:

Reconstitution of dehydrated media is done by dissolving a prescribed amount of the dehydrated media in a measured amount of distilled water. Reconstitution must be carried out using clean distilled water with a neutral pH value. The amount of dehydrated media to be measured in order to prepare a desired volume of nutrient media is prescribed by the commercial developer or distribute, along with sterilization and dissolving instructions. Dissolving dehydrated media in water should be carried out in laboratory grade glass containers, cleaned and rinsed with deionised water beforehand. Glassware should possess a volume three times larger than that of the medium prepared in them so as to avoid spillage and afford adequate dissolution. When dissolving the dehydrated media, ideally the solid media would be dissolved in half as much water before adding the remaining water. Thorough mixing is advised so as to create a homogenous mixture if possible before adding the other half of the water. This allows the interior walls of the glassware in question to be rinsed, as well as allows rinsing of any residues trapped on the laboratory spoon or the measuring vessel. Once the dehydrated media is properly suspended in water, appearing as a homogenous mixture, the media may be sterilized using an autoclave.

- Autoclave:

The release valve must be firmly closed before the heaters turn on. The pressure inside theautoclave is allowed to reach 0.5 bars or 0.5 atm (relative to atmospheric pressure). Afterwards the release valve is carefully opened ever so slightly as to allow a stream of steam to escape (characteristic noise). Once the pressure drops again, the release valve may be closed once more and the heaters allowed to reach 121 degrees centigrade. Generally the pressure may rise to 1 atm or even more, but as long as it remains within safety limits (generally 3 atm more than atmospheric), no sterilization issues should occur. Once the autoclave has reached 121 degrees centigrade, sterilization has commenced. Varying media have variable sterilization temperatures as well as pressures, however generally most media are designed to be sterilized at 121 degrees centigrade for 15 minutes under approximately 1 atm of additional pressure. The autoclave may be turned off once the sterilization time has elapsed. When the temperature drops to around 80 degrees centigrade, the release valve may be opened ever so slightly. Once the pressure drops, the autoclave should be generally safe to be opened.

- Pouring techniques for agar plates, agar slants, deep agar, test tubes (liquid media):

Media should be homogenized after sterilization and cooling and, as needed, should be reallocated (most often in Petri dishes). Pouring of agar based media should be carried out at 45-55 degrees centigrade with minimal water evaporation if possible. Pouring should be carried out in strict aseptic conditions, with particular care that the receiving container be sterile. Some media allow for redistribution to be carried out in test tubes or other glassware before sterilization. Pouring media onto Petri plates may be carried out in two different styles. One alternative is to measure 13-15 ml of the desired medium and to transfer those 13-15 ml onto a sterile plate. In which case, the test tube’s opening should be passed by an open flame or similar sterilization procedures should be used. The test tube cap would ideally be held by the pinky finger of the person carrying out the pouring. Once the contents of the test tube have been transferred to the plate, gentle circular movements should be carried out in order to homogenize the mixture further and to assure an even distribution of the medium. The Petri dish should lay on a flat surface near a flame or under other sterile conditions, and allowed to solidify. Plates containing solid media may be flipped once the media has solidified utterly, preventing further water condensation from impacting the homogeneity of the system, hindering colony observations or potentially liquefying cultured colonies. The second technique involves directly pouring media from the Erlenmeyer flasks they were sterilized in, onto sterile plates, with the volume of media being determined approximately by hand-eye coordination (13-15 ml). Pouring may result in bubble formation, which may be removed by applying the flame of a Bunsen burner near the plate. Solidified plates may be kept for a variable period of time depending on the amount of media, temperature of refrigeration, humidity etc. Generally most media are to be kept at 4-8 degrees centigrade. Solid media may be prepared as agar slants or deep agar as needed. Agar slants may be prepared by pouring a determined amount of sterile medium in a sterile test tube, and slanting the test tube on either a slant surface or a raised surface by resting the top of the test tube on the raised surface and the bottom of the test tube on the lower surface. The liquid media inside covers a larger surface area this way, and, once solidified, allows for a larger surface to be cultivated. Contamination must be carefully avoided by using test tube caps and keeping the test tubes in aseptic conditions if possible. The small space required by these test tubes makes agar slants ideal for conserving stock cultures and pure cultures previously isolated and purified. This conservation method in particular is highly effective, as risk of contamination is much lower than with Petri plates. Deep agar is prepared by allowing the poured media in the test tube to solidify upright, allowing cultivation of anaerobic microorganisms at the bottom of the deep agar. Liquid media are usually poured in test tubes, Erlenmeyer flasks or other glassware before sterilization. Test tubes of 16x160mm size should receive from 5 to 10 ml of respective medium, presenting as a column with height of about 4-8 cm, depending on the thickness of the glass etc.

Planned and performed by Sofija Kostandinovska, Institute of Biology, FNSM, Ss. Cyril and Methodius University, Skopje, Macedonia.