MICROBIOLOGY LAB MANUAL
Lab 3
Culturing of Microorganisms
Learning Objectives:
Following this exercise the student should be able to:
1.
explain the principles behind the use of solid and broth media for culturing.
2.
perform an environmental culture using the appropriate tools and techniques.
3.
identify and describe three different colony morphologies grown from their
specimen.
4.
interpret the significance of growth in a broth culture.
5.
determine the purity of a culture.
Bacteria are invisible to the naked eye. In order to study them we use two significant
techniques: 1) microscopy, in conjunction with staining to make them visible and 2)
culturing bacteria so that there will be large enough numbers to observe masses of
bacteria with the naked eye. A single bacterium multiplies by binary fission to produce a
characteristic mass of genetically identical bacteria. This mass is called a colony and is
considered to be genetically pure. The shape, consistency, color, margin (border), and
elevation of the colony are as characteristic of a species of bacteria, as the shape size
and color of a fish is to a species.
When grown in liquid media (broth culture) bacteria will cause the tube to look cloudy in
the region where growth is heaviest. Some bacteria form a pellicle (a thick layer) on the
surface of the fluid, others form cotton ball shapes, and some appear to have sunk to
the bottom of the tube as if sticking to the bottom. This growth relates to the bacteria's
oxygen requirements. The microbiologist cannot pick out individual bacterial types from
a broth. In order to separate and isolate different bacterial types they are grown on solid
media. This media has all the required nutrients, as the broth culture had, but a
solidifying agent, called agar, is added to the mixture. Solid media allows many
observations to be made about the colonial morphology of different bacteria. We use
solid media in tubes (agar deeps), in tubes that have been tipped and allowed to solidify
with a surface for growth (agar slant), and in plates called Petri dishes.
Agar slants (in tubes) are useful for maintaining bacterial growth over long periods of
time without drying. Petri dishes allow observation, easy access, and isolation; but are
prone to environmental contamination and desiccation.
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1
Investigative Assignment
Using the transport culture swab, select a location that you hypothesize may contain
many invisible microbes. Just before class, open the paper cover and using aseptic
technique* roll and rub the cotton swab on the area you want to culture. Place it back in
the sterile** plastic sleeve. Label the culture source and break the ampule at the bottom
of the container to release the transport medium that will nourish the microorganisms
until you subculture the specimen.
Dir. Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
___________________________________________
Surface selected for culturing
*Aseptic technique means free of contaminating organisms other than those you are
investigating. For example, you would not want your hand microbes to contaminate the
swab.
**Sterile means free from all life forms.
Lab Period 1Materials for each individual:
PPG (personal protective gear)
1 inoculated culturette swab
1 Tryptic Soy Agar (TSA) plate
1 Tryptic Soy Agar (TSA) slant
1 nutrient broth
transfer loop
gloves
sharpie
Materials for each lab team:
Test tube rack
small beaker with disinfectant
incinerator
Method:
The streak plate method is used to isolate individual colonies of bacteria for observation
and identification. After 24 hours of growth the final product should look like this picture.
This is tricky; the bacteria must be dispersed while they are still invisible to your eyes.
Proceed as follows placing a check in th
the end of each step after it is completed:
1.
2.
Label the tubes and plate with your initials,
the culture source, and the date. Read the Atlas on
Page
4.
Sterilize the loop, by holding it in the incinerator for 10 seconds. Remove the
loop, hold it as it cools. (This is composed of nichrome wire, a rapidly heating and
2
3.
Remove the swab from the plastic container.
Open the Petri dish placing the lid upside down on a
surface thereby preventing contamination of the lid and lab top. Without contacting the
swab to any other surfaces, roll the swab across quadrant number 1 of the agar plate,
the initial surface to be cultured. Recover and replace the Petri dish to limit
Dir. Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
5.
Carefully open the Petri dish, placing the lid upside down once again. Touch the
surface of the agar to gauge its durability. You want to rub the bacteria across the agar
without gouging it. Rub the loop gently over the surface of the medium at the very end
6.
7.
Sterilize the loop, allow it to cool. Make the final broad strokes through quadrant
8.
Inoculating the broth culture. Using the culturettes again, aseptically remove the
swab. Open the top of the nutrient broth; place the top of the tube near the incinerator (5
seconds). Insert the swab in the broth. Reheat the mouth of the tube. Close the tube
9.
Inoculating the agar slant. Using the culturettes again, aseptically remove the
swab. Open the top of the agar slant; place the top of the tube near the incinerator (5
seconds). Insert the swab and roll it up the slant. Reheat the mouth of the tube. Close
10.
Be sure everything is labeled. Incubate the plate (upside down). Place the agar
slant and broth culture in a test tube rack; put the rack and plate in the 37C incubator.
Check the temperature on the incubator. Be sure the doors are completely closed to
as
11.
12.
Place the contaminated material in the appropriate location at the biohazard
13.
Return other materials and equipment to the appropriate location, including the
14.
Lab Period 2 Materials for each individual:
small beaker with disinfectant
incinerator
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3
PPG (personal protective gear)
3 Tryptic Soy Agar (TSA) plate
transfer loop
gloves
sharpie
Materials for each lab team:
Dir. Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
1. Plug in the incinerator.
2. Put on your PPG and collect your plates for observation.
3. Examine the various colony morphologies present on your plate. * DO NOT ever
open any plate that has a white fuzzy growth on it – in Bakersfield this could be
the Valley Fever Fungus. Call the instructor if anything looks fuzzy.
4. Select 3 different types of colonies. Number them using your sharpie and writing
on the bottom of the plate (not the lid – it moves).
5. Review in the Atlas (pages 1-4) terminology for the macroscopic observations of
colonies on solid media. Refer to the table below for examples of typical terms
used to describe colonial morphology. Practice using these terms to describe
colonies to your lab partners.
For example this colony could be described as white,
raised, rhizoid, opaque. It is hard to tell in the picture
whether it is dry or moist.
These colonies would be
described as spreading, irregular,
moist, and gray.
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4
Colonies of Swarming Proteus
mirabilis© Shapiro, author. Licensed
for use, ASM MicrobeLibrary (linked
to http://www.microbelibrary.org)
Dir. Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
Colony Size
Punctiform
Small
Large
Borders
Circular
Common Descriptions for Colony Morphology
Size of colonies correlates to some species
Very small dots
.:.:
Small colonies are relative, of course, but colonies with a diameter
of 1-2 mm would be considered small
Regardless of the shape, colonies ranging over 8 mm would be
considered large
The outer edge of the colony
Round colonies with even borders
Filamentous
Fibrous looking, with stringy extensions
Irregular
Extending unevenly from the center with asymmetrical borders
Loboid
Border of colony appears to extend out in lobes
Rhizoid
The border of the colony appears to extend out like tree roots - see
picture and description above
The colony is shaped long and thin like a spindle
Spindle
Spreading or
swarming
Elevation
Flat
Raised
Umbonate
Pitted or concave
Texture
Shiny or Wet
Mucoid
Dry
Opaque
Color
Colored colonies
Depressed into the agar
The texture of the colony relates to it moistness or dryness,
whether you can see though it or it is opaque
Colony appears shiny and moist
Colony appears covered with mucous
Colony appears dry and sometimes folded
Colony is solid, an object behind the colony cannot be seen
Most colonies have some color - many are just shades of white or
grey, but the color is an important characteristic and tool in
identification
Some colonies have characteristic colors other than the color of the
agar such as: Orange, Yellow, White, Pink, Black, Red
As described above, this colony would appear clear, like glass, or a
drop of water
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5
Translucent
Edges of the colony appear wavy
See picture above
The elevation of the colony refers to the height or depth of the
colony compared to the plane of the agar
Colony seems even with the surface of the agar
Colony appears as a mound on the agar
Slightly raised in the center
Dir. Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
Lab Report for Exercise #3
Name _________________________ Culture Source________________________
1. Select 2 or 3 different colonies from your culture. Draw a sketch of each colony
below. Describe the colony on the chart below using the descriptive terminology
on the previous page. You may use the colony counter to get a better view.
Colony
Sketch
Description
#1
#2
#3
1. Subculture these organisms by transferring each of these bacteria to a new plate
using the isolation technique described in the Atlas (see page 9). These colonies
will be used to practice staining next lab hour.
Original environmental mixed culture
Streak for isolation
Colony #1
Colony #2
Colony #3
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6
Three pure cultures
Dir. Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
2. Observe the growth in your broth culture.
Draw a picture of the tube in the box to the
right indicating bacterial growth. Circle the
type oxygen requirement this growth
indicates:
strict aerobe
microaerophile
facultative anaerobe
strict anaerobe
mixed growth & requirements
3. List the advantages and disadvantages of a
broth culture compared to a solid culture.
4. Why is the culture incubated upside down?
5. What would occur if the lid of the Petri dish was placed right side down on a
tabletop?
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6. You are asked to use a culturette to collect a wound specimen. Would you
inoculate the specimen on to solid agar plate, a slant, or into a broth culture?
Explain why.
Dir. Janet Fulks
Bakersfield College
August 2010