Cultivation of Bacteria 1

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The Microbiology Laboratory & Cultivation of Bacteria

1.1 Introduction to Culture Media:
The study of microorganisms requires being able to grow them in the laboratory. Bacteria are grown in
culture media, which provides the nutrients, necessary for the organisms of interest. There are
different ways to classify the media:
1- Consistency:
Liquid: is called a Broth and it is usually placed in a test tube or a flask.
Solid: Liquid media that has been solidified by the addition of agar (usually 1.5 % w/v) a complex
polysaccharide. The solid media can be placed in Petri dishes (agar plates) or test tubes with a large
surface area (agar slants).
2- Type of media:
Nutrient media: is a specific chemical formulations that contain all the nutrients
and minerals that a many microorganisms needs for normal growth. It is called
defined when the specific nutrients and their amounts are known. An
undefined media is one where the exact composition is not known.
Selective media: is a type of media that favors the growth of a specific

microorganism over others. In fact others may be inhibited by media
components.
Differential media: permit the recognition of specific microorganisms. Usually

by taking advantage of some biochemical reaction that might produce a specific
change in the media (color is often used)
1.2 Aseptic Technique in Transferring Bacteria:

One of the first requirements to study specific microorganisms is to
separate them from the mixed microbial populations in which they are found in
the environment. To achieve this goal microbiologists use culture media and
aseptic transfer techniques.
To start, aseptic technique is used to introduce a very small sample of cells

(the inoculum) into a receptacle containing nutrient or culture medium. This
process is called inoculation.
The aseptic (sterile) technique is a technique designed to keep the

working environment as free of contaminants as possible. This is achieved first,
by sterilizing all equipment and media that will be in contact with the
microorganisms. This includes minimizing the air movement on the working area.
Usually the work is done within the vicinity of a flame. Aseptic technique is
required for the maintenance of pure cultures and the successful isolation of
specific types of microorganisms.
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A pure culture is a culture that contains only one species of bacteria. A

mixed culture encompasses more than one species. When isolating bacteria
from the environment the microbiologist always starts with a mixed culture. A
pure culture can be obtained from the mixed culture by sub-culturing and
streaking for isolation.
Use of the Loop/Stab Inoculator:

Two different types of inoculators can be used depending on the purpose of the
work. The loop is used to a) transfer cultures from one medium to another, b) to
prepare bacterial smears, and c) to streak plates. The loop is the tool of choice
for working with a liquid inoculum culture. The stab is used to prepare stab
cultures and to pick single colonies from a plate.
EXERCISE 1:
Inoculating Liquid Media

For this exercise a tube containing a liquid culture will be used to provide the
source of the inoculum for broth and agar slants. Work close to the flame!
1) Hold the source culture tube with your non-dominant hand and hold the
inoculating loop with your dominant hand. Carefully shake the culture to make
sure the cells are resuspended
2) Sterilize the inoculator by first passing the entire wire through the flame,
starting at the handle end. Wait until the whole wire becomes red hot. Allow
the wire to cool (without waving it on the air) for about 30 seconds.
3) Remove the cap from the source tube using the pinkie finger of the same hand
that is holding the inoculator. You will hold the cap in your finger until it is time
to put it back on the source tube.
4) Pass the mouth of the source tube through the flame once.
5) Without touching the walls of the tube, put the sterile inoculator into the
source tube containing the culture and dip the loop into the liquid.
(NOTE: If you were using a slant or plate as a source you will simply touch the
surface of the agar where the bacteria are growing with the inoculator. We will do
this in the near future.)
6) Withdraw the inoculator from the source tube, and replace the cap.
7) Remove the cap of the tube to be inoculated (using the pinkie finger as before)
and insert the inoculator into the tube. Don’t stir or shake the loop
excessively. Flame the top of the tube and replace the cap.
. (NOTE: See the specific instructions below for the different types of solid
media.)
8) Flame the inoculator as before but, heat it slowly so that any material
remaining on the loop does not spatter.
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Inoculation of an agar slant:
Rest the inoculator gently at the lower end of the slant and withdraw it slowly
upwards moving it from side to side (the surface of the agar should not be
broken). This should leave a streak on the surface of the slant (In some specific
experiments you may be require to stab the slant just under the agar surface, if
that is the case it will be clearly specified in the instructions).
Inoculation of an agar stab:

Using aseptic technique pick a single well isolated colony with a sterile
inoculating stab needle and stab the needle several times through the
agar to the bottom of the vial or tube. Replace and tighten the cap. Make
sure the tube and cap are well labeled. Give the stab to your instructor
for storage.
Inoculation of an agar plate:

Working with agar plates is bit different than working with media in tubes in that
you have a wide lid instead of narrow cap. This means there is a greater
surface area of sterile media that can be exposed to contaminations in the
atmosphere. The key is to keep as much of the lid over (covering) the
open agar plate as possible. Never set the lid down on the lab bench
when in an open contaminating environment. Who the agar in a plate is
inoculated depends on the goal. In the next exercise we will swap the
plate to get as many possible microbes across the surface as possible.
Next lab period we will try to isolate a single colony of a bacterium, in
which case we use the “streaking for isolation” technique.
1.3 Hunt Microbes from the Environment:
Fomites are inanimate objects, which carry viable pathogenic organisms. Equipment and
objects used by many individuals such as restaurant’s eating utensils, public phones, ATM machines,
and money are some of the common objects that may act as vehicles of dissemination for some
bacteria.
Public health microbiologists routinely examine utensils used in public eating establishments
and are able to assess the sanitary conditions prevailing based on the results obtained from such
examination. There are various procedures employed in the examination of utensils. One of these
employs a cotton swab soaked in a phosphate buffer. The swab is rubbed over the surface of the
object(s) under study, then returned to the test tube of buffer in which it was originally soaked. The top
of the swap is broken off into the buffer while the portion contaminated by handling is discarded.
Portions of the liquid from the tube containing the swap tip are plated on solid nutrient medium and
colony counts obtained indicate the degree of contamination present.
In this exercise we will use a slightly different method. You will use a sterile swab, which you
will rub over the surface to be tested, and then is streak over the surface of a plate containing nutrient
agar and a selective medium.
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Name_____________________ Date__________
Examination of fomites for bacterial contamination:

MATERIALS:
1. Sterile cotton swabs.
2. Tube of sterile distilled water.
3. Petri dishes with nutrient agar
4. Object to be tested : Your group will be assigned one of the following
a ATM machine versus Public Phone
b) Money (coins versus paper)
c) Bathrooms (male versus female)
d) Tables on cafeteria versus benches on the microbiology lab
Your instructor will give you specific instructions for each location.
PROCEDURE:
1. Carefully withdraw a sterile cotton swab from its container, making sure that the cotton is moisten
(but not too wet) with the sterile water.
2. Rotate the side of the swap, rather than the tip, over the portion of the object to be tested.
3. Smear and rotate the swap thus contaminated over the surface of the agar on the Petri dish.
4. Label the Petri dish to indicate the object tested.
5. Incubate the inoculate dish upside down at room temperature for three or more days.
6. In the next lab period you will be asked to exam the dish, count the colonies, and grade the level of
contamination as follows:
0 to 10 colonies/dish - Good (low contamination)

11 to 50 colonies/dish - Fair (some contamination)
Over 50 colonies/dish - Poor (heavy contamination)
7. Next lab period, also describe colony morphology for your two media types as indicated in Unit 1,
Exercise 2 (above).
8. Discuss which fomites were most contaminated and offer an explanation.
9. Discuss differences between media types.

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2.1 Bacterial Colony Characteristics:
A bacterial colony is a very homogeneous population formed by the

progeny of a single bacterium. The colony becomes visible with the naked eye
after several millions of individual are produced. Since all the members of the
colony are derived from a single cell, all the characteristics of the bacteria within
that population are essentially the same. Different bacteria (i.e. different species,
and sometimes strains) have specific colony characteristics on specific media.
Thus, evaluation of colony characteristics is one of the first steps in the process of
identifying bacteria. Always select a typical well isolated colony for your
observations. Crowded colonies are going to be small due to competition for
nutrients between adjacent colonies.
There are seven macroscopic characteristics of bacterial colonies

that must be observed and recorded. (Figure 1.3) These are:
1- SIZE: whenever possible measure colony diameter and express it in

millimeters mm. If the bacteria colony is too small to be measured you may
describe it as punctiform.
2- SHAPE: look at the whole colony. Describe the shape using the terms: circular,
irregular, filamentous, or spindle as indicated in figure 2.1.
3- MARGIN: Describe the outer edge of the colony as entire/smooth,
undulate/wavy, filamentous as indicated in figure 2.1
4- SURFACE: select one of (a) and one from (b)
a) smooth, wrinkled, rough, concentric rings
b) dull (matte), or shiny (glistening)
5- PIGMENTATION: Describe the color as precisely as possible

6- TRANSMITED LIGHT: hold the plate up to the light. Call it translucent if the
light passes through the colony or opaque if the light does not pass through
the colony
7- ELEVATION: hold plate to the side and note if the colonies are flat, raised,
convex, umbonated or craterform.
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Figure 1.3. Bacterial colony morphology characters and

descriptions.
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1

The Microbiology Laboratory & Cultivation of Bacteria

Selective media: is a type of media that favors the growth of a specific

Differential media: permit the recognition of specific microorganisms. Usually

1.2 Aseptic Technique in Transferring Bacteria:

To start, aseptic technique is used to introduce a very small sample of cells

The aseptic (sterile) technique is a technique designed to keep the

A pure culture is a culture that contains only one species of bacteria. A

Use of the Loop/Stab Inoculator:

Inoculating Liquid Media

Inoculation of an agar stab:

Inoculation of an agar plate:

1.3 Hunt Microbes from the Environment:

Examination of fomites for bacterial contamination:

4. Label the Petri dish to indicate the object tested.

0 to 10 colonies/dish - Good (low contamination)

8. Discuss which fomites were most contaminated and offer an explanation.

9. Discuss differences between media types.

2.1 Bacterial Colony Characteristics:

A bacterial colony is a very homogeneous population formed by the

There are seven macroscopic characteristics of bacterial colonies

1- SIZE: whenever possible measure colony diameter and express it in

5- PIGMENTATION: Describe the color as precisely as possible

Figure 1.3. Bacterial colony morphology characters and

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