E X E R CISE

6
MICROBIOLOGY

Ubiquity of Bacteria

Learning Outcomes
After completing this exercise, you should be able to
1. Culture bacteria from natural sources.
2. Appreciate that the kinds of bacteria and their
numbers will vary for the different environments
sampled.

Bacteria are the most widely distributed organisms

in the biosphere. The major portion of biomass on
the earth is made up from bacteria and other micro-
bial life. Bacteria occur as part of the normal flora of
humans and animals, where they occupy sites on sur-
faces such as the skin and intestinal tract. In humans it is
estimated that the normal flora outnumber our own cells Figure 6.1 Nutrient agar plate exposed to the
environment.
by approximately tenfold. In plants they are found on © Andreas Reh RF
leaf surfaces and in nodules on roots, where they form a
symbiotic partnership with the plant to fix nitrogen from billion (109). However, a colony can arise from more than
the atmosphere. They are also responsible for a variety one cell, for example when a chain of cells such as strep-
of diseases in both plants and animals. We depend on tococci grow on a nutrient medium. The cultivation of
bacteria to break down our sewage and waste. They are bacterial cells was vastly improved in Koch’s laboratory
important in the soil, where they are active in the nitro- when Frau Hesse, the wife of an early coworker of Koch,
gen and carbon cycles, thus contributing to soil fertility. suggested the use of agar-agar as a solidifying agent. A
Bacteria are abundant in the oceans, where they contrib- major advantage was that agar media were easily manip-
ute to cycles and mineralization. In the oceans, some ulated, and agar, unlike gelatin, was not degraded by the
have unique metabolic capabilities that allow them to pathogenic bacteria Koch was studying. As a solidifying
grow next to superheated hydrothermal vents and utilize agent, agar could be added to rich broths to form a solid
hydrogen sulfide and other gases emitted from volcanic medium on which isolated colonies would develop after
activity associated with these vents. Bacteria have been inoculation. The purity of cultures was further improved
isolated from core samples taken from deep within the when R. J. Petri, a worker in Koch’s lab, introduced a
earth’s crust. They form colorful blooms in sulfur hot covered dish that protected the nutrient surface of media
springs found in places such as Yellowstone National from contamination by bacteria in the environment, espe-
Park. In essence, they have been found in almost every cially the air. These methods were extremely important
place humans have searched for them. Thus, they are in the accomplishments of Koch’s lab because they pro-
almost ubiquitous in their distribution on the earth. vided a means for separating and culturing the pathogens
When Robert Koch first studied bacteria in his labo- that they were studying, including anthrax caused by
ratory, one of the first challenges was to devise a method Bacillus anthracis. Microbiologists still use agar and the
to grow bacteria in culture so that populations could be petri dish in the cultivation of bacteria today.
separated into individual species. Initial approaches used No single medium exists that will support the growth
pieces of vegetables such as potatoes and carrots, or the of all bacteria, owing to the diverse metabolic capabilities
addition of gelatin to meat broths. It was found that bac- and requirements of bacteria. However, many bacteria
terial cells would grow as visible, discrete colonies on a will grow on extracts of meat or vegetables that have
solid medium. A colony is a visible mass of cells usually been solidified with agar, an example being nutrient agar.
resulting from the division of a single cell (figure 6.1), During this laboratory period, you will be provided with
and the number of cells in a single colony can exceed one sterile bacteriological media that you will expose to the

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EXERCISE 6 Ubiquity of Bacteria

environment in various ways. Any bacterial cells that 3. Moisten a sterile swab by immersing it into a tube
occur in the environment and are deposited on the agar of nutrient broth and expressing most of the broth
medium or in the broth will subsequently undergo cell out of it by pressing the swab against the inside
division to produce colonies or cause the broth to become wall of the tube.
turbid. The idea is to appreciate that bacteria are ubiqui- 4. Rub the moistened swab over a part of your body
tous in the environment and can be spread by convection such as a finger or ear, or some object such as a
currents in the air. It is important to also understand doorknob or telephone mouthpiece, and return the
that the morphology of individual bacterial colonies that swab to the tube of broth. It may be necessary to
develop on an agar medium usually differ from one spe- break off the stick end of the swab so that you can
cies to another. This may involve different aspects of replace the cap on the tube.
the colony such as the regularity of its edge, pigmenta- 5. Label the tube with your initials and the source of
tion, the configuration and texture of its surface, and its the bacteria.
elevation. Examples of these characteristics are given in 6. Expose the blood agar plate by coughing onto it.
Exercise 35, figure 35.4 on page 250. Label the bottom of the plate with the initials of
You will be provided with three kinds of sterile bacte- the individuals who cough onto it. Be sure to date
riological media that you will expose to the environment the plate also.
in various ways. To ensure that your exposures cover as 7. Incubate the plates and tube at 37ºC for 48 hours.
wide a spectrum as possible, specific assignments will
be made for each student. This may involve the use of Evaluation
a swab to remove bacteria from an object or surface; in
other instances a petri plate containing a nutrient medium After 48 hours’ incubation, examine the tube of nutri-
will be exposed to the air in different environments. A ent broth and two plates. Shake the tube vigorously
number will designate your assignment, as detailed in without wetting the cap. Is it cloudy or clear? Com-
the chart below. pare it with an uncontaminated tube of broth. What is
the significance of cloudiness? Do you see any colo-
Materials nies growing on the blood agar plate? Are the colonies
all the same size and color? If not, what does this indi-
per student: cate? Group together a set of TSA plates representing
• 1 tube of nutrient broth all nine types of exposure. Record your results on the
• 1 petri plate of trypticase soy agar (TSA) Laboratory Report.
• 1 sterile cotton swab Your instructor will indicate whether these tubes
• Sharpie marking pen and plates are to be used for making slides in Exer-
per two or more students: cise 11 (Simple Staining). If the plates and tubes are to
• 1 petri plate of blood agar be saved, containers will be provided for their storage
1. Scrub down your desktop with a disinfectant (see in the refrigerator. Place the plates and tubes in the
Exercise 8, Aseptic Technique). designated containers.
2. Expose your TSA plate according to your assign-
ment in the chart below. Label the bottom of your Laboratory Report
plate with your initials, your assignment number,
and the date. Record your results in Laboratory Report 6.

EXPOSURE METHOD FOR TSA PLATE STUDENT NUMBER

7 Ubiquity of Bacteria
1. To the air in laboratory for 30 minutes 1, 10, 19, 28

2. To the air in room other than laboratory for 30 minutes 2, 11, 20, 29

3. To the air outside of building for 30 minutes 3, 12, 21, 30

4. Blow dust onto exposed medium 4, 13, 22, 31

5. Moist lips pressed against medium 5, 14, 23, 32

6. Fingertips pressed lightly on medium 6, 15, 24, 33

7. Several coins pressed temporarily on medium 7, 16, 25, 34

8. Hair combed over exposed medium (10 strokes) 8, 17, 26, 35

9. Optional: Any method not listed above 9, 18, 27, 36

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 6
Student: ______________________________________________

Laboratory Report Date: _________ Section: ______________________________

6 Ubiquity of Bacteria

A. Results
1. After examining your TSA and blood agar plates, record your results in the following table and on a simi-
lar table that your instructor has drawn on the chalkboard. With respect to the plates, we are concerned
with a quantitative evaluation of the degree of contamination and differentiation as to whether the organ-
isms are bacteria or molds. Quantify your recording as follows:
0 no growth
 1 to 10 colonies    51 to 100 colonies
  11 to 50 colonies     over 100 colonies
After shaking the tube of broth to disperse the organisms, look for cloudiness (turbidity). If the broth
is clear, no bacterial growth occurred. Record no growth as 0. If the tube is turbid, record  in the last
column.

PLATE EXPOSURE METHOD COLONY COUNTS BROTH

STUDENT
INITIALS
TSA Blood Agar Bacteria Mold Source Result

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Ubiquity of Bacteria (continued)
Use the class results to answer the following questions.
2. Using the number of colonies as an indicator, which habitat sampled by the class appears to contain the
most bacteria? ______________________________________________________________________
3. Why do you suppose this habitat contains such a high microbial count? _________________________
______________________________________________________________________________________
4. a. Were any plates completely lacking in colonies?
_____________________________________________________________________________________
b. Do you think that the habitat sampled was really sterile?
_____________________________________________________________________________________
c. If your answer to b is no, then how can you account for the lack of growth on the plate?
_____________________________________________________________________________________
d. If your answer to b is yes, defend it:
_____________________________________________________________________________________

B. Short-Answer Questions
1. In what ways do the macroscopic features of bacterial colonies differ from those of molds?
___________________________________________________________________________________
2. Why is the level of contamination measured as number of colonies rather than size of colonies?
___________________________________________________________________________________
3. Should one be concerned to find bacteria on the skin? How about molds? Explain.
___________________________________________________________________________________
4. How can microbial levels be controlled on the skin? On surfaces in the environment? In the air?
___________________________________________________________________________________
5. Compare the following features of bacteria to those of eukaryotic microorganisms:
a. size.
___________________________________________________________________________________
b. organization of genetic material.
___________________________________________________________________________________
c. ribosomes.
___________________________________________________________________________________
d. cell wall.
___________________________________________________________________________________
e. respiration and photosynthesis.
___________________________________________________________________________________
f. motility mechanisms.
___________________________________________________________________________________

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