Exercise 8Culture Media

Once the microscopic morphology and staining characteristics of a microorganism present in a clinical specimen are known, the microbiologist can make appropriate decisions as to how it should be cultivated and what biological properties must be demonstrated to identify it fully.

First, a suitable culture medium must be provided, and it must contain the nutrients essential for the growth of the microorganism to be studied (see Exercise 2). Most media designed for the initial growth and isolation of microorganisms are rich in protein components derived from animal meats. Many bacteria are unable to break down proteins to usable forms and must be provided with extracted or partially degraded protein materials (peptides, proteoses, peptones, amino acids). Meat extracts, or partially cooked meats, are the basic nutrients of many culture media. Some carbohydrate and mineral salts are usually added as well. Such basal media may then be supplemented, or enriched, with blood, serum, vitamins, other carbohydrates and mineral salts, or particular amino acids as needed or indicated.

In this exercise, we will prepare a basic nutrient broth medium and also a nutrient agar from commercially available dehydrated stock mixtures containing all necessary ingredients except water. The term nutrient broth (or agar) refers specifically to basal media prepared from meat extracts, with a few other basic ingredients, but lacking special enrichment. We will also see how liquid and agar media are appropriately dispensed in flasks, bottles, or tubes for sterilization before use, and how a sterile agar medium is then poured aseptically into petri dishes.

Purpose

To learn how culture media are prepared for use in the microbiology laboratory

Materials

Dehydrated nutrient agar

Dehydrated nutrient broth

A balance, and weighing papers

A 1-liter Erlenmeyer flask, cotton plugged or screw capped

A 1-liter glass beaker

A 1-liter graduated cylinder

Glass stirring rods (at least 10 cm long)

10-ml pipettes (cotton plugged)

Test tubes (screw capped or cotton plugged)

Petri dishes

Aspiration device for pipetting

Procedures

1. Read the label on a bottle of dehydrated nutrient agar. It specifies the amount of dehydrated powder required to make 1 liter (1,000 ml) of medium. Calculate the amount needed for 1/2 liter and weigh out this quantity.

2. Place 500 ml of distilled water in an Erlenmeyer flask. Add the weighed, dehydrated agar while stirring with a glass rod to prevent lumping.

3. Set the flask on a tripod over an asbestos mat. Using a Bunsen flame, slowly bring the rehydrated agar to a boil. Stir often. An electric hot plate may be used instead of a Bunsen burner.

Morello-Mizer-Granato: I I. Basic Techniques of I 4. Cultivation of I I © The McGraw-Hill

Laboratory Manual and Microbiology Microorganisms Companies, 2003 Workbook in Microbiology,

Figure 8.1 Preparing a plate of agar medium by pouring melted sterile agar into it.

Figure 8.1 Preparing a plate of agar medium by pouring melted sterile agar into it.

Aseptic Technique Pouring

4. When the agar mixture is completely dissolved, remove the flask from the flame or hot plate, close it with the cotton plug or cap, and give it to the instructor to be sterilized in the autoclave.

5. While the flask of agar is being sterilized, prepare 500 ml of nutrient broth, adding the weighed dehydrated powder to the water in a beaker for reconstitution and dissolution.

6. Bring the reconstituted broth to a boil, slowly. When fully dissolved, remove from flame or electric burner and allow to cool a bit.

7. The instructor will demonstrate the use of the pipetting device. Do not pipette by mouth. Using a pipette, dispense 5-ml aliquots of the broth into test tubes (plugged or capped). The instructor will collect the tubes and sterilize them.

8. When the flask of sterilized agar is returned to you, allow it to cool to about 50°C (the agar should be warm and melted, but not too hot to handle in its flask). Remove the plug or cap with the little finger of your right hand and continue to hold it until you are sure it won't have to be returned to the flask. Quickly pour the melted, sterile agar into a series of petri dishes. The petri dish tops are lifted with the left hand, and the bottoms are filled to about one-third capacity with melted agar (fig. 8.1). Replace each petri dish top as the plate is poured. When the plates are cool (agar solidified), invert them to prevent condensing moisture from accumulating on the agar surfaces.

9. Place inverted agar plates and tubes of sterilized nutrient broth (cooled after their return to you) in the 35°C incubator. They should be incubated for at least 24 hours to ensure they are sterile (free of contaminating bacteria) before you use them in Exercise 9.

Results

After at least 24 hours of incubation at 35°C, do your prepared plates and broths appear to be sterile?

Record your observation of their physical appearance:

Broths:

Morello-Mizer-Granato: I I. Basic Techniques of I 4. Cultivation of I I © The McGraw-Hill

Laboratory Manual and Microbiology Microorganisms Companies, 2003 Workbook in Microbiology, 7/e

Morello-Mizer-Granato: I. Basic Techniques of 4. Cultivation of © The McGraw-Hill

Laboratory Manual and Microbiology Microorganisms Companies, 2003

Workbook in Microbiology, 7/e

Questions

1. Define a culture medium.

2. Discuss some of the physical and chemical factors involved in the composition, and in the preparation, of a culture medium. Nutrient ingredients:_

pH and buffering:

Heat (to reconstitute): Heat (to sterilize):

Other:

3. At what temperature does agar solidify? _

At what temperature does agar melt?

4. What would happen to plates poured with agar that is too hot?

Could they be used? 5. What would happen to plates poured with agar that is too cool? Could they be used?

Morello-Mizer-Granato: I I. Basic Techniques of Laboratory Manual and Microbiology

Workbook in Microbiology, 7/e

6. Why are culture media sterilized before use?

7. Discuss the relative value of broth and agar media in isolating bacteria from mixed cultures.

8. Are nutrient broths and agars, as you have prepared them, suitable for supporting growth of all microorganisms pathogenic for humans? Explain your answer.

Morello-Mizer-Granato: I I. Basic Techniques of I 4. Cultivation of I I © The McGraw-Hill

Laboratory Manual and Microbiology Microorganisms Companies, 2003 Workbook in Microbiology, 7/e

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Responses

  • tekle
    What is the relative value of broth and agar media?
    8 years ago
  • Tanja
    What would happen to plates poured with agar that is too hot?
    7 years ago
  • damian
    Are nutrient broths and agars, as you have prepared them, suitable for?
    7 years ago
  • Ulrich
    Are nutrient broths and agars, as you have prepared them, suitable for supporting?
    8 years ago
  • petronio loggia
    Are nutrient broths and agar, as you have prepared them, suitable for?
    8 years ago
  • amethyst proudfoot
    What would happen to plates poured with agar that is too cool?
    8 years ago
  • theobald
    What is the relative value of broth and agar media in isolating bacteria from mixed cultures.?
    8 years ago
  • Monte Saladino
    Why are culture media steralized before use?
    8 years ago
  • piia
    How using aseptic technique for pouring agar plates?
    8 years ago
  • LAKESHA
    Are broths and agars suitable for growth for all microorganisms pathogenic to humans?
    8 years ago
  • sampsa
    Why use heat to reconstitute culture medium?
    8 years ago
  • Ishbel
    Why use heat to reconstitute culture media?
    8 years ago
  • haile
    Is agar suitable for supporting growth of all microorganisms pathogenic for humans?
    8 years ago
  • primula underhill
    Are nutrient broths and agar suitable for supporting growth of all pathogenic in humans?
    8 years ago
  • letizia fiorentino
    Are nutrient broths and agars suitable for growth all microorganisms pathogenic for humans?
    8 years ago
  • mebrahtu
    Do your prepared plates and broth appear to be sterile?
    8 years ago
  • wiseman
    Are nutrient broths and agars suitable for supporting growth of all?
    8 years ago
  • eyob
    Why are culture media sterilised before use?
    8 years ago
  • Dieter
    Are nutrient broths and agars suitable for supporting growth of all microorganisms?
    8 years ago
  • keiran
    How to prepare 500 ml nutrient broth media?
    8 years ago
  • Casimiro
    What are some of physical and chemical factors involved in a culture medium?
    8 years ago
  • astolfo
    What is the chemical factoe and the preparation of a culture medium involving nutrient ingredients?
    7 years ago
  • frodo
    What are chemical and physical factors involved in compotion and prepration of culture medium?
    7 years ago
  • GANDOLFO
    Are nutrient broths and agars suitable for supporting growth of all pathogens of humans?
    7 years ago
  • alessia genovese
    Which components of a culture medium are important for pH and buffering (Morello 53)?
    7 years ago
  • Kirsi
    Are nutrients broths and agars suitable for growth of all microorganisms?
    7 years ago
  • kenneth
    What are the physical and chemical factors involved in a cultural medium?
    7 years ago
  • Maisy
    What happened to sterilized plates and broths?
    6 years ago

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