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EXERCISE 1

PREPARATION OF AGAR SLANT, NUTRIENT BROTH AND AGAR PLATE

The cultivation of microorganisms in vitro, apart from its substrate is possible by providing
substances that the organisms can use as food. Preparations containing the needed substances are called
culture media. Media are classified according to: 1) composition, 2) consistency and 3) selectivity.
Media classification according to composition can either be empirical or natural (e.g. peptone,
vegetable infusions) and synthetic or defined (organic or inorganic). Based on consistency, media are
classified as liquid (broth), solid (slant, plate) and semi-solid (soft agar). According to selectivity,
media are categorized as general or non-selective (e.g. nutrient agar) enrichment or selective (e.g.
lactose broth) and differential (e.g. EMB agar).
Sterilization is required to render a medium or material free of all forms of life. The most useful
approach is autoclaving, in which the items are sterilized by exposure to steam at 121˚C and 15 psi of
pressure for 15 minutes. Modern autoclaves are designed to ensure that all of the air has been expelled
and only steam is present in the autoclave chamber.
The survival and growth of microorganisms depend on available and favorable growth
environment. For the successful cultivation of a given microorganism, it is necessary to understand its
nutritional requirements and then supply the essential nutrients in the proper form and proportion in a
culture medium. Using solid media in Petri plate, one can observe the growth of different
microorganisms from a sample through spread plating method, isolation of microorganism into pure
culture can be done through streak plating method and the cultural characteristics of microorganisms
can be observed. In addition, different biochemical tests for pure cultures of microorganisms can be
carried out using solid media in Petri plates.

OBJECTIVE
To familiarize students with the process of media preparation and operation of a sterilizer or an
autoclave
To learn how to prepare pre-poured agar plates
To become familiar with the technique of pouring media in Petri plates

MATERIALS
Commercially prepared nutrient agar (NA) slant for bacteria
Commercially prepared nutrient broth (NB) for bacteria
Pipettes
Glass syringe
Test tubes and screwcap tubes
Aluminum foil or wax paper
Distilled water
Flasks
Cotton plugs
Pre-sterilized Petri plates
Wax Paper or Weighing Paper
Beaker & stirring rod Flasks
Rubbing Alcohol

PROCEDURE (SLANT & BROTH)


1. Calculate the total amount of medium needed. Decide how many slants and broths are needed.
2. Cut out a piece of aluminum foil or wax paper large enough to hold the amount of powdered medium
needed.
3. Using a dry spatula, weigh the amount of medium needed. Avoid unnecessary exposure of the
medium to the atmosphere because of its hygroscopic nature.
4. Fold the foil or wax paper at once and label with marking pen.
5. Carefully transfer weighed powder into flasks or Gatorade bottles. Wash off any sticking powder
with distilled water into the flask.
6. Add required volume of distilled water, swirling the flask or bottle to mix after each addition.
7. Melt the agar in microwave oven
8. For making slants, dispense around 7 ml melted agar per test tube using a 10 ml pipette or a glass
syringe and place the cotton plug. For making broth, dispense around 5 ml of the melted medium into
the tubes.
9. Put test tubes together and cover with aluminum foil or place in autoclavable plastic so as to protect
cotton plugs from getting wet which may subsequently cause contamination.
10. The culture media should be sterilized right away.
11. After sterilization, lay sterile test tubes containing agar medium across slanting boards and allow
them to solidify. Let the sterile broth tubes cool completely.
12. Refrigerate the sterile media for future use.

PROCEDURE (PLATE)
1. Calculate the total amount of medium needed. To make one plate, 15 to 20 ml is required.
2. Cut out a piece of weighing paper large enough to hold the amount of powdered medium needed.
3. Using a dry spatula, weigh the amount of medium needed. Avoid unnecessary exposure of the
medium to the atmosphere because of its hydroscopic nature.
4. Fold the weighing paper and label at once with marking pen.
5. Carefully transfer weighed powder into flasks of Gatorade bottles. Wash off any sticking powder
with distilled water into the flask.

6. Add required volume of distilled water, swirling the flask or bottle to mix after each addition. No
need to melt the agar using microwave.
7. Sterilize using the autoclave.
8. Pour the sterile medium into pre-sterilized Petri plates. The technique will be demonstrated by the
lab instructor.
9. Gently tilt from side to side or put the plate down and move it in a back-and-forth, right-left direction
to distribute the agar uniformly over the bottom. Do not let the medium come into contact with the
cover.
10. Allow the medium to solidify and invert the plates to prevent condensing moisture from
accumulating on the agar surface. All plates should be incubated for at least 24 hours to ensure sterility
before you use them.

QUESTIONS
1. Differentiate the various types of media based on composition, consistency and selectivity.
2. What is agar? Why is it a good support for microbial growth? Why is gelatin not often used in the
preparation of culture media in a microbiology laboratory?
3. What is the difference between agar and gelatin in terms of properties like chemical composition,
temperature required for melting and temperature required for solidification?
4. Why is the agar in test tubes allowed to solidify in a slanted position?
5. Why is it necessary for culture medium to be cooled to about 48 ˚- 50 ˚C before it is poured into
Petri plates?
6. What is the purpose of flaming the Petri plates before and after dispensisng the medium?
7. What are some ways to prevent contamination of culture media in Petri plates?