Methods of Culturing
Microorganisms
Specimen Collection
Five basic techniques
1. Inoculate
2. Incubate
3. Isolation
4. Inspection
5. Identification
Inoculation
•Culture: the propagation of microorganisms with various media
•Medium (pl. media): a nutrient used to grow microorganisms
outside their natural habitat
•Inoculation: the implantation of microorganisms into or onto
culture media
Incubation
Incubator: media containing inoculants are placed in temperaturecontrolled chambers
•Usual laboratory propagation temperatures fall between 20°C and 40°C
•Atmospheric gases such as O2 and CO2 may be required for the growth
of certain microbes
•During incubation, microbes grow and multiply, producing visible
growth in the media
Isolation
•Based on the concept that if
an individual cell is separated
from other cells on a nutrient
surface, it will form a colony
•Colony: a macroscopic cluster
of cells appearing on a solid
medium arising from the
multiplication of a single cell
Discussed in more detail
later in notes
•Requires the following
- a medium with a firm
surface
- a Petri dish
- inoculating tools
Inspection
Identification
Isolation: A single visible colony represents a pure culture or
single type of bacterium isolated from a mixed culture
Isolation: Various Conditions of Cultures
Pure Culture
(a)
One type of
microbe
Mixed Culture
(b)
More than one
type of microbe
Contaminated Culture
(c)
Unwanted
microbe present
Question
• Can you isolate a colony using liquid
culture?
Three basic methods of
isolating bacteria
• Streak Plate
• Loop Dilution
• Spread Plate
Isolation: Streak Plate
Isolation: Loop Dilution
Isolation: Spread Plate
Media
• Classified according to three properties
– Physical state
– Chemical composition
– Functional types
Physical State
• 1. Liquid media (I.e. tryptic soy broth=TSB)
• 2. Semi-solid media
• 3. Solid media (I.e. tryptic soy agar=TSA)
17
Liquid media are water-based solutions that are generally termed
broths, milks and infusions.
Semi-solid media contain a low percentage (<1%) of agar, which
can be used for motility testing.
Solid media contain a high percent (1-5%) of agar, which enables
the formation of discrete colonies.
Chemical content
• Synthetic media
• Nonsynthetic or complex media
Chemically defined media
- media whose compositions are precisely chemically
defined
- contain pure organic and inorganic compounds that vary
little from one source to another
•
molecular content specified by an exact formula
Minimal media
- contain nothing more than a few essential compounds
such as salts and amino acids
-
some contain a variety of defined organic and
inorganic chemicals
Complex media
contain ingredients
that are not
chemically defined
or pure (i.e. animal
extracts).
TSA + 5% sheep blood
(Do we know the exact
percentages of chemicals in
the sheep blood?)
23
Functional types of growth
media
• Selective media
• Differential media
Comparison of Selective and Differential Media
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Mixed
sample
Mixed
sample
General-purpose
nonselective medium
(All species grow.)
Selective medium
(One species grows.)
(a)
General-purpose
nondifferential medium
(All species have a similar
appearance.)
(b)
Differential medium
(All 3 species grow but may
show different reactions.)
Selective media enables (selects) one type of bacteria to grow,
while differential media allows bacteria to show different
reactions (i.e. colony color)
Selective media
enables (selects)
one type of
bacteria to grow,
while differential
media allows
bacteria to show
different reactions
(i.e. colony color)
Differential media we
will use in class
Mannitol salt agar (MSA)
contains 7.5% salt which
selects for gram positive
and also contains mannitol
(carbohydrate) plus a pH
indicator to indicate when
mannitol has been
fermented to an
acid…media turns from
pink to yellow
Differential media we will use in class
MacConkey agar contains bile salts and dyes that inhibit grampositive bacteria and hence selects for gram negative. Also turns
dark color when lactose is fermented
28
Maintenance and Disposal of Cultures
•Cultures and specimens constitute a potential hazard
•Prompt disposal is required
•Stock cultures represent a “living catalog” for study
and experimentation
•The American Type Culture Collection (ATCC) in
Manassas, VA is the largest culture collection in the U.S.
Microscopy
Science relies on technology
• The progress of microbiology and many other
disciplines relies heavily on the introduction of
new technologies
• Technologies such as new types of media or
microscopes lead to advances in microbiology
• Without a microscope it would be impossible to
characterize the morphology of bacteria.
History of the Microscope
• Development of the microscope originated in
1676 by Anton van Leeuwenhoek
• Luckily, he was a skilled observer and artist
and produced many fine illustrations of
microbes (“animalcules”)
• Unfortunately, he was not willing to share his
microscopes or knowledge with other
scientists until much later in life to help the
progress of microbiology
We measure specimens based on the
metric system. We will be looking at
bacteria ranging from 1 um to 10 um.
33
Principles of Light Microscopy
•Magnification
- objective lens: closest to the specimen, forms the
initial image called the real image
-
ocular lens: forms the second image called the
virtual image that will be received by the eye and
converted to the retinal and visual image
•Total magnification
Power of Objective
Usual power of
ocular
Total magnification
4x scanning objective
10x
40x
10x low power
objective
10x
100x
40x high dry objective
10x
400x
100x oil immersion
objective
10x
1000x
Magnification is useless without
good resolution.
•Resolution (resolving power)
- the capacity of an optical
system to distinguish or
separate two adjacent points or
objects from one another
- the human eye can resolve
two objects that are no closer
than 0.2 mm apart
• The shorter the wavelength, the better the resolution (the
ability to see 2 separate units)
The Effect of Wavelength on Resolution
(a)
Low resolution
(b)
High resolution
High resolution distinguishes magnified objects clearly
Wavelength and Resolution
• Is (a) or (b) resolved?
• Which resolves images
better, gamma rays or
visible light?
Properties of Light
A, B, & C
D
• Light has the ability to a) reflect, b) transmit (pass
through), c) absorb, d) refract and
Resolution can be increased by using immersion oil.
A compound microscope is typically used in teaching and
research laboratories
One objective lens can hold up to a
dozen small lenses inside
Optical microscopes
• All have a maximum magnification of
1000x--2000X
–
–
–
–
–
–
Bright-field
Dark-field
Phase-contrast
Differential interference
Fluorescent
Confocal
Types of Optical Microscopes
Bright-field
• Most commonly used in laboratories
• Observe live or preserved stained specimens
Types of Optical Microscopes
Dark-field
• Observe live unstained specimens
• View an outline of the specimens
Types of Optical Microscopes
Phase-contrast
• Observe live specimens
• View internal cellular detail
Fluorescent Microscopy
• Fluorescence stain or dye
• UV radiation causes emission of visible
light from dye
• Diagnostic tool
Fluorescent staining on a fresh sample of cheek
scrapings from the oral cavity. Fuzzy cells are
cheek cells, distinct rods and cocci are bacteria.
Red=dead, green=live
Example of a confocal microscope
Fluorescence or
unstained specimen
images are
combined to form a
three-dimensional
image.
Electron microscopy
• Very high magnification (100,0001,000,000X)
• Transmission electron microscope
(TEM)
– View internal structures of cells
• Scanning electron microscope (SEM)
– Three-dimensional images
Transmission
Electron
Microscopy (TEM)
Example of
False-color
Scanning
Electron
Microscopy
(SEM)
Comparison of optical and electron microscopes
What does an electron
microscope use
to visualize specimens?
Which type of
microscope has better
resolving power, brightfield or electron?
In lab, we use Bright-Field Light Microscopy
• We will perform the following light microscopy
techniques:
• Wet mounts
– Wet mount and hanging drop methods
• Smears
– Spread, air-dry, heat fix
• Simple Staining
– Cheek cells with methylene blue (BI231)
• Differential Staining
– Gram stain
– Acid-fast (time permitting)
• Special Staining
– Capsule stain
– Flagellar stain (prepared slides since this is a difficult technique)
– Endospore stain (time permitting)
Wet mounts
• We will use the hanging drop
technique to investigate
bacterial motility:
– Advantages of the hanging
drop:
• Easy to prepare
• Bacteria are live so we can see
motility
–Disadvantages of the
hanging drop:
•Requires special
depression slide
•Difficult to visualize
since microbes are not
stained
•Bacteria are live and
therefore slides must be
disinfected
Smears
• Performing quality smears takes practice. There are many
steps in the preparation of a smear that must be performed
correctly. (Think of Goldilocks that wants her porridge “just
right”)
• 1. Smear bacteria onto slide from broth or agar
• Too thick and you can’t see through specimen, too thin and you
can’t find any bacteria
• 2. Air dry
• Again, you do not want too large a smear or it will take a long time
for the smear to completely air dry.
• 3. Heat Fix (see next slide)
Smears (cont.)
• 3. Heat Fix
• Pass slide through flame quickly 3-4 times
• Heat fix too little and organisms may wash off slide
• Heat fix too much and organisms may be distorted
• Heat fixation:
– 1. kills organisms
– 2. adheres specimen to slide
– 3. promotes stainability of specimen
Stains
• Positive stains
– Dye binds to the specimen
• Negative stains
– Dye does not bind to the specimen, but
rather around the specimen.
Which type of stain do we usually use?
Why do positive stains bind to the specimen?
What makes a specimen negatively charged?
Positive stains are basic dyes
(positive charge) that bind
negative charge cells, and
negative stains are acidic dyes
(negative charge) that bind the
background.
Molecular structure of
methylene blue
(a positive stain attracted to ?)
60
Positive stains
Simple stain
– One dye
• Ex. Methylene blue, crystal
violet
• In BI231, you applied one
stain (methylene blue) to
visualize your cheek
(buccal) cells.
Positive stains
Differential stain
– Two-different colored dyes
• Ex. Gram stain
• Allow us to differentiate between
types of bacteria.
– Gram Stain
• Gram-positive vs. Gram-negative
• See next slide
– Acid-Fast Stain
• Used to identify acid-fast bacteria with
a waxy, lipid cell wall.
• Detects Mycobacteria like M.
tuberculosis and M. leprosae
• Most of the bacteria in our lab in AcidFast negative
Positive Stains:
Gram Stain
• Most frequently used stain
• Dictates which type of antibiotic
treatment
• Gram-Positive
–
–
–
–
Purple
Penicillin
Thick Peptidoglycan
4 P’s (Positive, Purple, Penicillin,
Peptidoglycan)
– 1 plasma membrane
• Gram-Negative
–
–
–
–
Red
Streptomycin
Thin peptidoglycan
2 plasma membranes
63
Positive stains
Special stain
– Emphasize certain cell parts
• Ex. Capsule stain
Special Stains
We use these special stains to identify special structures
that some bacteria possess
• Capsule Stain
• Capsule does not stain so we stain the background
• Can not heat-fix or capsule will be destroyed
• Flagellar Stain
• Very difficult stain, we will look at prepared slides
• Endospore Stain
• The endospore is difficult to stain since it is made of a
thick, hard to penetrate spore wall. We will boil the stain
to help it penetrate the spore coat.