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Laboratory Exercise 4 CULTURE MEDIA PREPARATION

The survival and continuous growth of microorganisms depend on adequate supply of nutrient and favorable environment. A culture medium is any nutrient material prepared for the growth and cultivation of microorganisms. It must satisfactorily supply all the factors required by the microorganisms for growth. The chemical compounds may be grouped as:

1. Nitrogen sources

2. Carbon sources

3. Energy sources

4. Minerals

5. Growth factors

The medium that is used to culture the microorganisms depends on the microorganism that one

is trying to isolate or identify. Different nutrients may be added to the medium, making it higher in protein

or in sugar. Various pH indicators are often added for differentiation of microbes based on their biochemical reactions; the indicators may turn one color when slightly acidic, another color when slightly basic. Other added ingredients may be growth factors, NaCl, and pH buffers which keep the medium from straying too far from neutral as the microbes metabolize. The culture medium can be prepared from basic ingredients or from commercially available digests. It can exist in three consistencies: liquid, solid, and semisolid. A liquid medium lacks solidifying

agent and is called broth medium. Agar is a component used for solidifying bacteriological media. It is

a gel-forming polysaccharide which is extractable from several species of red species (agarophytes). It

is a mixture of at least two polysaccharides: agarose (ca. 70%) and agaropectin (ca. 30%), with D- galactose and 3,6-anhydro-L-galactose as chief components. Agar liquefies at 100°C and solidifies at 40°C. Agar should be capable of remaining molten and fluid at 40-45°C after cooling from boiling point. Different grade of agar are available and depending upon the brand, the required amount of agar will vary. A completely solid medium requires an agar concentration of 1.5% to 1.8%. A concentration of less than 1% agar results in semi-solid medium.

In this experiment, each student will:

1. Compare the different forms of culture media in terms of function and composition; 2. Prepare different culture media.

MATERIALS / EQUIPMENT:

Used bond papers Petri dishes (2 per person) 10ml culture tubes (4 per person) alcohol lamp Media bottles (2 per group) 2 Test tube racks (metal and wooden) masking tapes/autoclave tapes 150-200 ml Erlenmeyer flasks (2 per group) Labelling tapes

PROCEDURE:

10mL pipettes cotton, gauze, and white thread Autoclave hot plate Nutrient Agar (NA) Nutrient Broth (NB) Eosin-Methylene Blue (EMB) Agar

Preparation of cotton plugs

Plugs made of non-absorbent cotton wool are used in test tubes, flasks, and pipettes to prevent micro-organisms from passing in or out and contaminating either the culture or the environment. The necessary movements of air in and gaseous products out are not prevented and the gaps between the cotton wool fibers are even wide enough for micro-organisms to pass through. However, this does not happen because micro-organisms (negatively charged) are “filtered” out by being attracted to and adsorbed on the oppositely charged cotton wool. The cotton wool must remain dry because this filtration property is lost if the

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cotton wool becomes moist hence the use of non-absorbent cotton wool.

1. Place the gauze on the mouth or edge of a flask or test tube. Use your finger to press into the center of the gauze to allow space for the cotton wool. Make about 1 cm space allowance for test tubes and about 3 cm for flasks.

2. Tightly stuff the gauze with cotton wool until it reaches the edge of the tube/flask.

3. Use a thread to knot and tighten the gauze. Leave 3 cm of gauze as a handle.

4. Test the cotton plug by pulling out the handle.

5. The gauze should be re-used twice

Preparation of the NA and EMB

1. Most of the basic microbiological media is available in powdered or dehydrated form. Since most of the stock media are in the dehydrated form, avoid too much exposure of the media to the environmental moisture. Keep the media container tightly closed. Always use a dry spatula in obtaining the media.

2. Calculations for the amount of media powder in proportion to distilled water will be demonstrated in class by your Instructor. One may also check for the instruction label of microbiological media for media preparation.

grams of NA powder or EMB powder and

3. Culture media is prepared by weighing out

dissolved to

ml of distilled water. Be careful not to get this powder on the balance.

4. Before turning on the thermal stirrer, one should have hand towels or potholders handy. Using the towels to protect your hand, grasp the neck of the flask tightly and remove it from the stir plate.

5. The next step will require application of heat to the mixture. Before doing this, however, one should be aware that agar has a strong tendency to boil when it reaches 100°C. DO NOT USE EXCESSIVE AND SUDDEN RISE IN TEMPERATURE TO AVOID CHARRING OF THE MEDIUM. Use minimum amount of heat during sterilization. To avoid over boiling during sterilization, do not fill the vessel with more than 2/3 full of liquid.

6. At first sign that the mixture is boiling and the agar is dissolved, it will turn clear, deeper tan for NA. For EMB, it will turn reddish purple with a greenish cast.

7. Turn off the thermal stirrer, take the flask off the heat and allow it to cool for a few minutes.

8. Label the flask with the media name, date, and Family Names of the members of the group.

9. After preparation of the media, place the flask in the autoclave with those of the rest of the class. Follow Procedure B for sterilization.

10. After sterilization, allow the culture media to cool down. Store the media in a cool, dark place and in a tightly stoppered container (to avoid contamination) in the refrigerator and not near the freezer area. Plate-dispensed media are only good for 1 week provided they are free of contamination.

Preparation of the Agar Slants and Agar Deeps/Stabs

1. Dissolve the dehydrated medium in distilled water (Check label instructions).

2. Heat to boiling on a medium temperature to facilitate proper dissolution and dispersion of the medium particles.

3. Allow the medium to cool a bit and dispense or pipe out 5 ml into test tubes.

4. Cover the SLANT tubes and the DEEP tubes with clean cotton plugs. Sterilize the medium in an autoclave.

5. Place all of the tubes which have been pipetted out in the test tube rack.

6. SLANTS are prepared by solidifying the agar with the tube in a slanting position (the size and the angle of the slant will determine whether it is a regular or a special slant).

7. BUTTS are prepared by solidifying the tube in an upright position.

8. If the media are not used immediately; store them in a ziplock or plastic container and place in the refrigerator. Always label the containers with the name of the media and the date of preparation.

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Preparation of the Broth

1. Dissolve the dehydrated broth medium in distilled water (Check label instructions).

2. Heat to boiling on a medium temperature to facilitate proper dissolution and dispersion of the medium particles.

3. Allow the medium to cool a bit and dispense 5ml into clean and dry 10 ml culture tubes.

4. Cover the tubes with cotton plugs and sterilize the medium in an autoclave. Tighten the plugs after sterilization.

5. If the media are not used immediately; store them in a ziplock or plastic container and place in the refrigerator. Always label the containers with the name of the media and the date of preparation. No heat need be applied at this stage.

Study Questions:

Short but concise answers must be placed after every question. Copy each question first before writing your answer. Maximum of 2 sentences.

1. List the factors that will guide you in choosing what media to prepare.

2. Why do we always use distilled water instead of tap water in dissolving our media?

3. What are the problems encountered in media sterilization? What could have been the cause of such problem?

4. Classify the different culture media according to:

a. Composition

b. Consistency

c. Manner of preparation

d. Function