Micro-Para Laboratory

Department of Biological Sciences

Institute of Arts and Sciences
Far Eastern University

LABORATORY EXERCISE 6
Bacterial Cultivation

As you learned in Lab 3, microbes exist everywhere, and very rarely do

they occur as a single species. Robert Koch, known as the father of
medical microbiology, was one of the first to recognize that isolating a
microbe (in his case, bacterium) away from other microbes was crucial
for his own argument that microbes cause disease, as well as
understanding characteristics of the microbe itself. His studies on
Bacillus anthracis contributed to many of the laboratory techniques we
still use today, including the method for isolating pure cultures of
bacteria.

The most used method in the laboratory for isolating microbes is the
streak plate, and to a lesser
extent, the pour plate. Both methods rely on dilution of bacterial cells in a sample to the point at which a single cell can
divide giving rise to a single pure colony. The pure colony is essentially a clone of cells that all are identical copies of
the original cell and can be used for further study.

Protocols for use of cultivation of bacteria, use of general growth, enriched, selective and differential media,
plate pouring, determination of temperature range for growth and determination of salt tolerance capacity of bacteria.
In nature, bacteria exist as mixed populations. In the laboratory these populations must be separated so that
characteristics of individual species may be observed. Several basic techniques are used in microbiology to obtain
pure cultures; some involving a single step from the initial culture others require a series of purification steps to ensure
that only the targeted organisms are obtained. Whatever method is chosen, it is important to observe the aseptic
techniques in handling of all materials and in doing all the manipulations called for.
First, microorganisms must be removed from natural environments and cultured in the laboratory. This
requires artificial media and surfaces on which bacteria may grow. This also requires knowledge of nutritional
requirements and environmental requirements (such as temperature of incubation and the requirement of oxygen).
Second, bacteria of interest must be separated from all other bacteria in the environmental sample. This
requires separation techniques that allow isolation of a pure culture of one type of bacteria.
Third, once a pure culture is achieved, no contaminating bacteria can be introduced from the environment.
This requires that all media and laboratory supplies be sterile (that is contain no bacteria that may contaminate the
culture of interest).
Fourth, techniques are needed that facilitate working with pure cultures. This requires aseptic technique and
techniques of storage for pure cultures.
When microbial ecologists seek to isolate new bacteria from the environment, they must experiment with
many nutrients and growth conditions to culture the newly isolated bacteria in the laboratory. It is often very difficult to
replicate bacterial growth conditions in the laboratory. It is estimated that only 0.1% of all bacteria have been
successfully cultured.
A. Types of media
The fundamental function of bacterial media is to provide nutrients for the growth of microbes in the laboratory.
Media designed for growth and the different types are:
1. General Growth media

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

2. Chemically Defined media

3. Enriched media (fastidious)
4. Selective media
5. Differential media
B. Solid vs Liquid
Organisms can be grown in liquid media (broth) or on solid media. For example, Nutrient media is referred to
as Nutrient Broth when in the liquid form, and Nutrient Agar when in the solid form. Agar, a galactan obtained from
marine algae is used as the hardening agent in solid media. Broth, with added agar powder, is heated to 121 oC in an
autoclave, dissolving the agar and sterilizing the medium. The molten medium may be poured into plates or tubes. The
agar-media will remain liquid at temperatures above 45oC. Below 45oC the agar will harden and supply a solid surface
for the growth of bacteria. Agar plates and slants may be inoculated after they have solidified. To avoid any
condensation-droplets from falling onto agar surfaces and smearing the inoculum, agar plates are inverted during
incubation.

Figure 1. Media constituents

Nutrient Agar: example of a complex, general growth media
Constituent Amount
Peptone 5.0g
Beef extract 3.0 g
Sodium Chloride 8.0 g
Agar 15.0 g
Water 1 1iter

Glucose Minimal Salts Broth: Example of chemically defined medium designed for the growth of E. coli. Constituent
Amount
Glucose 5.0 g
Ammonium phosphate monobasic 1.0 g
Sodium chloride 5.0 g
Magnesium sulfate 0.2 g
Potassium phosphate, dibasic 1.0g
Water 1 liter

Reference: Tortora, Funke, Case. (2004). Microbiology, An Introduction, 8th edition, Pearson Education, Inc. publishing as Benjamin Cummings

To study bacteria from an environmental sample or a mixed culture it is necessary to isolate a pure culture of
each bacterium of interest. Using differing growth conditions and a variety of media, one can begin to separate bacteria,
but, generally, methods to physically separate bacteria are also needed.

C. Use of Physical Separation Procedures:

a) Streak Plate technique
As we discussed in previous labs, single colonies may be achieved by using the streak plate technique.
Additional methods used to physically separate bacteria include dilution in liquid, the pour plate (where diluted cultures
are poured onto a solid medium spreading over its surface) and the spread plate (where diluted cultures are spread
onto a solid medium and thus distributed over the medium surface).

D. Growth Conditions
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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

To grow bacteria in the lab, environmental conditions, as well as nutrients, must be considered. Bacteria may
be isolated from a variety of environments. For cultivation of bacteria in the lab, the conditions of the environments
must be mimicked. Prokaryotes that live in extreme environments are generally in the Domain: Archaea. Those that
live in more moderate environments are generally in the Domain: Bacteria.

Consider Temperature.
Microorganisms whose optimal temperature for growth is near 0oC are called psychrophiles. Those that
grow optimally between 20C and 45C are called mesophiles. Those growing best at high temperatures, 55C to ~
70C and higher, are called thermophiles. Prokaryotes that live at very high temperatures (up to 113C) are
hyperthermophiles.

Consider salt tolerance.

Some bacteria require relatively high concentrations of salt for growth (10-20%); these organisms are called
halophiles. Halophiles must possess special membranes and enzyme mechanisms to survive in salty environments.
Some organisms are salt tolerant. They do not require salt for growth but may grow in its presence. An example is
Staphylococcus aureus. S. aureus is found on skin, which often has a high salt concentration (10% NaCl).

Consider the requirement for oxygen.

An organism that requires oxygen for growth is called aerobic. An organism whose growth cannot occur in
the presence of oxygen is anaerobic. An organism that can grow under either aerobic or anaerobic conditions is a
facultative anaerobe. Microaerophilic organisms require free oxygen but at a very limited concentration.

The requirement for oxygen relates to the energy metabolism of an organism and the presence of enzymes
that destroy toxic products of oxygen. Aerobes use respiration as a mechanism for energy metabolism, and oxygen
serves as the terminal electron acceptor. Aerobes have enzymes such as catalase that help the organism deal with
toxic forms of oxygen. Anaerobes do not use oxygen for energy metabolism and have no enzymes to cope with toxic
forms of oxygen. Facultative anaerobes do not require oxygen for energy metabolism, but many will use it if it is
present. They have enzymes to cope with toxic forms of oxygen. Because the requirement for oxygen relates so closely
to the energy metabolism of an organism, the requirement for oxygen will be investigated in the next laboratory activity.

Learning Objectives:
To investigate the requirement of media constituents and environmental conditions in the cultivation of bacteria.
• Learn to complete all protocols effectively.
• Learn to recognize positive and negative results for growth on general growth, enriched, selective
and differential media
• Learn to Pour plates, streak plate, swab method procedure
• Learn how to determine environmental requirements for bacterial growth
• Learn an appreciation for culture characteristics of bacteria.

Protocol 1: PREPARATION OF SOLID MEDIA:

Plate Pouring MATERIALS/group.
1 Sterile bottle containing 75ml Nutrient Agar (melted, autoclaved, in water bath at 60oC)
3 sterile petri plates Jar of powdered Nutrient Agar

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

METHOD
Preparation of Nutrient Agar.
• Dissolve the dehydrated medium in the appropriate volume of distilled water i.e., 28gm
dehydrated nutrient agar in 1000 mL distilled water.
• Heat with frequent agitation and boil for 1 minute to completely dissolve the powder
• Sterilized the medium by autoclaving (121°C for 15 min)
• Dispense the medium into tubes (i.e., 3 ml to make nutrient agar slopes, 5 ml to make nutrient
agar deeps) or plates. Left the agar medium to solidify.

Preparation of Mannitol Salt Agar.

• Suspend 111.025 gm of dehydrated media in 1000 ml of distilled water.
• Boil to dissolve the media completely.
• Autoclave at 121°C for 15-20 minutes.
• Cool to 45-50°C and pour into petri dishes.

Preparation of MacConkey Agar.

• Suspend 49.53 grams of dehydrated medium in 1000 ml purified/distilled water.
• Heat to boiling to dissolve the medium completely.
• Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
• Cool to 45-50°C.
• Mix well before pouring into sterile Petri plates.

Protocol 2: Bacterial cultivation

Streak plate method, spread plate method, swab method, and pour plate method
Agar plate (Nutrient Agar, Potato Dextrose Agar)
Three sterile plates of agar plates (NA, PDA)
Note: check the microbial sampling method

Materials
Agar plates (NA, PDA)
Mixed culture
Alcohol lamp
Inoculating loop, Sterile Cotton swab, L-rod shape
Petri plates, Test tubes, pipette (1 mL), aspirator
Parafilm

Protocol: Streak plate method

1. Prepare lab bench by removing extraneous items and cleaning surface with table disinfectant.
2. Label the bottom surface of your sterile agar plates.
3. Using the T-method (illustrated below) or quadrant method, outline the sections on the bottom of the agar plate.
4. Obtain mix culture and shake gently to suspend organisms.
5. Flame the loop until it is red-hot and let cool.
6. Remove cap of mix culture and flame the mouth of the tube (do NOT place cap down on the table).
7. Insert the loop into the tube and remove a loopful of broth with bacterial cultures.

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

8. Lightly flame the mouth of the culture tube again.

9. Return the cap to the tube and set in your tube rack.
10. Streak the plate, following either the T-method or quadrant streak shown below. Do not gouge into the medium
with the loop and keep the lid over the plate as much as possible.
11. Flame the loop before setting it down.
12. Repeat the above protocol (# 4-11) using your body broth from Lab 5 and a new agar plate.

Step 1: Do the initial inoculation then FLAME the loop, let cool about 15 seconds.

Step 2: Use the sterile cooled loop and draw over the agar surface in the first section, flame and cool.

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

Step 3: Use the sterile cooled loop and draw of the agar surface in the second section. Note that you do NOT go back
to the broth, and you overlap with the first section a few times. Flame the loop and allow to cool.

Step 4. Use the sterile cooled loop and draw of the agar surface in the third section. Again, do NOT go back to the
broth and overlap with the second section only. Now flame loop. Now place the plate in incubator.

13. Incubate the plates inverted at 30° C for 2 days. (NOTE: we will have to set up a separate 30° C
incubator for this experiment. Follow instructions on where to put these plates). See animation at:
http://www.sumanasinc.com/webcontent/animations/content/streakplate.html

Protocol: Pour plate method

1. Label the bottom of three sterile Petri plates I, II, and III.
2. Remove 3 tubes of nutrient agar from the 45 to 50 C water bath in the back of the room. (NOTE:
you must work quickly at this point, or the nutrient agar will solidify in the tube).
3. Using aseptic technique, take one loopful from the mixed culture and add to the nutrient agar in
tube I.
4. Mix the tube by rolling vigorously between the palms of both hands. DO NOT shake the tubes as the
caps are not secure and medium will splash out.
5. Immediately, and using aseptic technique, take a loopful of the agar from tube I and add it to tube II.
6. As soon as this is done, you may pour the contents of tube I into the plate labeled plate I, being sure
to pour it into the bottom of the plate.

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

7. Repeat steps 4-6, transferring a loopful of tube II to tube III, and then pouring the contents into
plates II and III.
8. Allow media to solidify and then incubate the plates inverted at 30° C for 2 days.
(NOTE: we will have to set up a separate 30° C incubator for this experiment. Follow instructions on
where to put these plates).

Figure 5. Pour plate method

Protocol: Spread plate method

1. Make a dilution series from a sample.
2. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of
an agar plate.
3. Dip the L-shaped glass spreader into alcohol.
4. Flame the glass spreader (hockey stick) over a Bunsen burner.
5. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully
rotating the Petri dish underneath at the same time.
6. Incubate the plate at 37°C for 24 hours.
7. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate
dilution factor to determine the number of CFU/mL in the original sample.

Figure 6. Spread plate method

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

LABORATORY EXERCISE 6

Group No.: Date Submitted:

Surname/s of members (List you names alphabetically)

RESULTS AND DISCUSSION

1. Compare your results from the streak-plate, spread plate, and pour-plate methods. Which method achieved the
best separation of species?
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2. Regarding bacterial growth on solid media, define the term “colony”.

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3. What advantage does the streak-plate method have over the pour-plate method?

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4. Before inoculating and pouring molten nutrient agar into a plate, why must the agar first be cooled to 45 to
50C?

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

Data and Observation

1. Evaluation of streak plates: Show within the circle the distribution of the colonies on your streak plate. Did you get
clear isolation of mixed culture? (Note: Photo or draw the image)

2. Evaluation of pour plates: Show the distribution of colonies on the plate I, II or III.

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 Micro-Para Laboratory
Department of Biological Sciences
Institute of Arts and Sciences
Far Eastern University

3. Evaluation of spread plates: Show the distribution of colonies on the spread plate I, II or III.

Table 1. Different methods of cultivation with colony growing in plates

Methods No. of Distinct colonies Predominating colonies

Streak plate method

Pour plate method

Spread plate method

CONCLUSION
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