Media Components & Inoculation

COMPONENTS Culture media are formulated to create appropriate environments for specific microorganisms. Some common media constituents include:

Amino-Nitrogen Peptones, hydrolysates, infusions, and extracts are the main sources of nitrogen in culture media, and are essential for microorganism reproduction and metabolism.

Growth Factors Sheep, horse and rabbit blood, serum, and vitamins are added to support or enhance microorganism growth.

Energy Source Carbohydrates and alcohols are added as carbon and energy sources, which stimulate growth of microorganisms. Carbohydrates also are used to aid in microorganism identification and differentiation.

Buffering Agents Phosphates, acetates, and citrates maintain the pH in culture media.

Minerals Phosphate, sulfate, magnesium, calcium, manganese, and iron salts provide trace elements needed for organism growth.

Selective Agents Antimicrobials, dyes, and bile salts are used to restrict the growth of certain organisms, while permitting the growth of others.

Indicator Dyes Dyes, such as phenol red and bromthymol blue, are used in the preparation of differential and selective culture media.

Gelling Agents Agar and gelatin are added to a liquid medium to change the consistency to a solid or semisolid medium.

  INOCULATION

Streak Method for Agar Plates The streak plate primarily is used for isolating microorganisms in pure cultures from specimens or samples containing mixed flora. Obtaining isolated colonies on plates allows colonial morphology and hemolytic reactions to be examined, and biochemical/serological testing to be performed.

1. With a sterile inoculating loop, streak a loopful of the sample across the surface of an agar plate. The four-quadrant streak is the most common, and accomplished by streaking and rotating the plate in four sections, one quarter at time, slightly overlapping the original streak area. The fourth quadrant contains the greatest dilution of microorganisms, and usually provides isolated colonies for further testing.
2. Incubate plates under favorable growth conditions.
3. Examine plates for isolated colonies.

Spread Plate Technique The spread plate technique is used for enumerating microorganisms.

1. Drop 0.1 mL aliquots from serial dilutions onto the surface of an agar plate.
2. Aseptically spread inoculum across the surface using a bent glass rod or sterile inoculating loop. By spreading the suspension over the plate, a dilution gradient is established to provide isolated colonies.
3. Incubate plates agar inverted in appropriate conditions.
4. Count colonies and calculate the number of microorganisms in the original suspension.

Pour Plate Technique The pour plate technique also is used for enumeration of microorganisms in a particular sample. In this technique, test samples or suspensions of microorganisms are mixed with molten agar (45–50°C). The agar is allowed to solidify, trapping the bacteria at separate discrete positions within the matrix of the medium. Although the medium holds bacteria in place, it is soft enough to permit growth of bacteria and the formation of discrete isolated colonies.

1. Perform serial dilution of sample.
2. Aseptically pipette microorganism dilutions into labeled petri dishes.
3. Add melted agar that has been cooled to approximately 44–45°C.
4. Mix well by slightly rotating plate with bacteria and agar mixture.
5. Allow the agar to solidify, trapping bacteria at separate discrete positions within the medium.
6. Incubate plates in a favorable environment.
7. Count the number of colonies and calculate the number of microorganisms in the original sample.