English: Cultivation of microorganisms: Inoculation and incubation

- Inoculation of test tubes from test tube samples:

The two test tubes are to be kept next to each other, with as much visibility of the contents of the test tubes as possible. The tubes are to be kept at a slight slant, reducing airborne contamination risk. The inoculation loop should be manoeuvred by the other hand. The test tube caps should be removed using the other hand, by grasping one cap with the pinkie finger and the other with the ring finger. The inoculation loop and the caps should be held simultaneously if possible. The test tubes should pass an open flame or other sterilization methods should be employed to further reduce risks of contamination. In the case of a liquid media, touching the sterile inoculation loop should be sufficient to acquire enough inoculum to successfully subcultivate. In the case that the second media be liquid as well, allowing the loop carrying the inoculum to simply rest in the liquid should be enough to guarantee inoculation should the inoculum be viable. The openings of the test tubes should pass a flame or other sterilization method before being closed. The inoculation loop must be sterilized before proceeding.

- Inoculation of agar slants from agar slants samples:

Repeating as before, the inoculum to be transferred is to be acquired by gently touching the sterile inoculation loop on the slant surface where visible growth may be observed. The inoculum should be applied in a zig-zag streak across the sterile slant.

- Inoculation of deep agar:

Deep agar is generally inoculated with a bacteriological needle, by effectively piercing the needle along with the inoculum into the deep agar. The test tube should be held in a vertical position while performing such manoeuvres, with the opening facing downwards as to minimize airborne contamination potential.

- Inoculation of agar plates by pour plate technique:

The pour plate technique is based on pouring the media at a relatively cool temperature (45-50 degrees centigrade) over the liquid inoculum transferred beforehand from the test tube or flask containing the microbial suspension onto an empty Petri dish awaiting the relevant medium. Homogenization is achieved by gentle circular movements of the plate after pouring an appropriate amount of medium. Thorough homogenization is achieved by first moving the plate front to back, then left to front, and finally in circles, with each movement repeated for as many as 15-20 times.

- Inoculation of agar plates by modified pour plate technique:

Alternatively, the liquid inoculum may directly be transferred in the test tube containing the medium to be poured and homogenization may be achieved by gently rotating the test tube between the palms of the technician. The volume of the medium in the test tube should not occupy more than half the test tube so as to allow efficient homogenization.

- Inoculation of agar plates by spread plate technique:

Solid plates may be inoculated by applying a liquid inoculum on the surface of the plate and spreading it with a sterile L stick or equivalent spatula. Spreading the inoculum should be carried out until the inoculum is spread across the entire surface designed for cultivation (usually the entire surface of the solid medium). The plate should not be moved until the liquid has had enough time to be absorbed by the medium.

- Incubation of inoculated cultures:

Ambient parameters, such as temperature, oxygen availability, humidity etc. have a deterministic effect on the development of microorganisms. Incubators and water baths serve to achieve the optimal temperature for the incubation of the culture of interest. Thermostats, or incubators, generally are comprised of a metal chamber with double walls with distilled water between them as a a thermal buffer. Heaters warm the water to the desired temperature. Control is maintained through a system of thermo-regulators, such as contact thermometers or other similar equipment. The temperature variance of different incubators is contingent on their make and quality, however in most cases, incubators should not show larger variance than +/- 1 degree centigrade. Temperature variance among water baths should not vary greater than +/- 0.1 degree centigrade. Water, as a highly effective thermo-conductor with a large thermal capacity, meets such requirements. Some incubators have appropriate valves, allowing ventilation and humidity regulation, or enable installing additional dishes or electrical appliances, located around the installation facility around the incubator. In order to prevent water loss during incubation, plates are to be kept inverted with the surface facing downwards. Prolonged incubation (several days) requires an additional open container of water to be present in the thermostat in order to maintain the desired degree of relative humidity.

RESULTS: Bacterial growth in the test tubes containing liquid broth can be observed by the change in turbidity of the medium. Bacterial growth on agar slants may be observed as dense colonies along the slant. Bacterial growth in deep agar may be observed as a column of visible growth. Bacterial growth on agar plates may be observed as dense colonies of the bacterium.

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