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Culture Media

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9 views29 pages

Culture Media

Uploaded by

rahat.pharmacy
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Culture Media

• Culture Medium: Nutrients prepared for


microbial growth
• Sterile: No living microbes
• Inoculum: Introduction of microbes into
medium
• Culture: Microbes growing in/on culture
medium
Agar

• Complex polysaccharide of marine alga


• Used as solidifying agent for culture media in
Petri plates, slants, and deeps
• Generally not metabolized by microbes
• Liquefies at 100°C
• Solidifies ~40°C
Culture Media

• Chemically Defined Media: Exact chemical


composition is known
• Complex Media: Extracts and digests of
yeasts, meat, or plants
– Nutrient broth
– Nutrient agar
In complex media
•The energy, carbon, nitrogen and sulfur
requirements are provided by proteins.
•Proteins are large, relatively insoluble
molecules produced by few microorganisms.
•Partial digestion by acids or enzymes reduces
protein to amino acids called peptones that
can be digested by most bacteria.
•Vitamins and other organic growth factors are
provided by meat/yeast extracts.
Culture Media

Table 6.2 & 6.4


Anaerobic Culture Methods

• Anaerobes might be killed in oxygen


• Reducing media
– Contain chemicals (thioglycollate or oxyrase)
that combine O2
– Heated to drive off O2
Anaerobic Culture Methods
• Anaerobic jar
• The atmosphere
of container has
less than 5%
oxygen, about
18% carbon
dioxide and no
hydrogen
• Each Petri
plates(oxyPlate)
is anaerobic

Figure 6.5
An Anaerobic Chamber
 Large scale
apparatus
 Filled with inert
gases like about
85% Nitrogen,
10% Hydrogen
and 5% Carbon
dioxide
 Equipped with air
locks to introduce
cultures and
materials
Special culture techniques
Capnophiles require high CO2
• Candle jar • CO2-packet
• Intestinal/respiratory • Activated by
tract crushing or
• low oxygen high CO2 moistening with
water
Biosafety Levels

• Level 1: No special precautions


• Level 2: Lab coat, gloves, eye protection
• Level 3: Biosafety cabinets to prevent
airborne transmission
• Level 4: Sealed, negative pressure
– The Hot Zone
– Exhaust air is filtered twice
– HEPA filters
– Space suits
Biosafety Level 4 (BSL-4)
Laboratory

Figure 6.8
Selective Media
• Suppress
unwanted
microbes and
encourage
desired microbes.
• Bismuth sulfite
agar….isolate
typhoid bacterium
from feces
• SDA for fungi
Figure 6.9b, c
Differential Media
• Make it easy to distinguish colonies of
different microbes.

Figure 6.9a
Enrichment Media
• Encourages growth of desired microbe
• Assume a soil sample contains a few
phenol-degrading bacteria and thousands of
other bacteria
– Inoculate phenol-containing culture medium with
the soil and incubate
– Transfer 1 ml to another flask of the phenol
medium and incubate
– Transfer 1 ml to another flask of the phenol
medium and incubate
– Only phenol-metabolizing bacteria will be
growing
• A pure culture contains only one species
or strain
• A colony is a population of cells arising
from a single cell or spore or from a group
of attached cells
• A colony is often called a colony-forming
unit (CFU)
Streak Plate

Figure 6.10a, b
Preserving Bacteria Cultures

• Deep-freezing: -50°to -95°C


• Lyophilization (freeze-drying): Frozen (-
54° to -72°C) and dehydrated in a vacuum
Reproduction in Prokaryotes

• Binary fission
• Budding
• Conidiospores (actinomycetes)
• Fragmentation of filaments
Direct Measurements of Microbial
Growth
The growth of microbial population can be
measured as cell number or population’s total
mass, which is directly proportional to cell
number.

Population numbers are usually recorded as


the number of cell in milliliter of liquid or in a
gram of solid material.

Direct and indirect methods of measurement

Serial dilution
PLATE COUNT
• The most frequently used method
• Advantage: measures viable cells
• Disadvantage: time taking at least 24 hrs
• Assumes each live bacterium grows and
divides to a single colony
• A colony results from short segments of
chain or from a bacterial clump.
• Plate counts are reported as colony
forming units (CFU)
• 25 to 250 or 30 to 300 are countable
• Plate Counts: Perform serial dilutions of a
sample

Figure 6.15, top portion


Plate Count

• Inoculate
Petri
plates
from
serial
dilutions

Figure 6.16
Plate Count
• After incubation, count colonies on plates
that have 25-250 colonies (CFUs)

Figure 6.15
Direct Measurements of Microbial Growth
• Filtration

Figure 6.17a, b
Direct Measurements of Microbial Growth

• Multiple
tube MPN
test
• Count
positive
tubes and
compare
to
statistical
MPN
table.
Figure 6.18b
Direct Measurements of
Microbial Growth
• Direct Microscopic Count
• Specially designed slide called Petroff-Hausser cell counter
• 0.01 ml sample spread over a marked square centimeter of slide
Direct Measurements of
Microbial Growth

Figure 6.19
Estimating Bacterial Numbers by Indirect
Methods
• Turbidity

Figure 620
Estimating Bacterial Numbers
by Indirect methods

• Metabolic activity (production of an acid


or carbon dioxide)
• Dry weight (for filamentous organisms)
• Fungus is removed from medium,
filtered and dried in desiccator and
weighed.

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