Transcript Culture media

Dilution calculations
1. You are interested in determining the number of bacteria in saliva.
You spit into a tube, and then do four 1/10 dilution's. From each
dilution tube you plate 200 ul onto an appropriate medium, and
observe 157 countable colonies on the agar surface after overnight
growth. How many bacteria are present in the original sample?
2. A friend of yours tells you that there should be no bacteria in
hamburger meat, and having had micro you say not true. To show
him/her you do the following: You take 1 gram of meat and blend it
in 100 ml of sterile water. You then do the following dilution: 1/10,
followed by 1/10 followed by 1/100. You then take 0.1ml from the
last dilution, and plate onto an appropriate medium, and find that
after 18 hours of growth that there are 125 colonies on the plate.
How many bacteria were present in the original sample, per ml of
blended material and per gram of hamburger meat?
3. You take a sample from your bacterial culture at 3h post inoculation.
You do the following dilutions: ¼, followed by 1/6, followed by 1/8
and then you place 169ul. After 24h you observe 125 colonies. How
many CFU/ml did you have in your sample at 3h post inoculation?
1.
You are interested in determining the number of bacteria in saliva. You spit into a tube, and then do
four 1/10 dilution's. From each dilution tube you plate 200 ul onto an appropriate medium, and
observe 157 countable colonies on the agar surface after overnight growth. How many bacteria are
present in the original sample
10-1 10-2 10-3 10-4
1ml transferred
9 ml in each test tube
200ul spread on each plate
Colonies
Plate 1
500
plate 2
350
plate 3
157
plate 4
15
CFU/ml= # colonies X DF X 1/volume plated
CFU/ml= 157 X 103 X 1/0.2 = 7.85 X 105
e.g. Hemocytometer calculation
You do a hemocytometer count and find you have 35 cells per primary
square. If you only did a 1/100 dilution of your culture sample before
you put it on the hemocytometer how many cells/ml do you have in
your culture?
Cells/ml= Average # cells in primary square X DF X 10 4
= 35 X 100 X 10 4
= 3.5 X 10 7 cells/ml
Culture media
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solutions containing all of the nutrients and
necessary physical growth parameters
necessary for microbial growth.
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CHONPS
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not all microorganisms can grow in any given
culture medium
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many can't grow in any known culture
medium.
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In addition to chemical and physical
characteristics, media can be distinguished
qualitatively as:
solid vs. broth
non-synthetic vs. chemically defined
selective
Differential, enriched
Solid medium
• media containing agar or some
other, mostly inert solidifying agent.
• has physical structure , allows
bacteria to grow in physically
informative or useful ways
• solid culture media immobilize
cells, allowing them to grow and
form visible, isolated masses
called colonies
• is usually used as: slants , stabs ,
petri dishes
Broth medium
• media lacking a solidifying matrix
Different kinds of media are used:
1. Non-synthetic [chemically undefined] medium , Chemically
undefined ingredient:
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contains at least one component that is neither purified nor
completely characterized nor even completely consistent from
batch to batch.
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May be broth or solid.
2. Chemically defined [synthetic] medium
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prepared from purified ingredients and therefore whose exact
composition is known.
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Not trivial for fastidious organisms:
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must know exactly what your microorganism's growth
requirements are.
If not known it must be discovered through trial and error and
therefore is not a trivial process for fastideous microorganisms.
3. Preprepared medium
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Media of many types can be obtained premixed, in an often
dehydrated and powdered state.
Sterilization
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Media must be sterilized before use, or cultures will
always have contaminants
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Autoclaving- heating the medium to a temperature of
121°C, under 110 kPa (~ one atmosphere) of steam
pressure above ambient.
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Filtration- some media components can not be
sterilized by heating , can filter through small pore
filters
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Dry, or wet and volume are all issues considered when
determining how long to autoclave for.
Aseptic technique- ensuring you only grow
the organism you want to grow!
• Successful cultivation and maintenance of
pure cultures of microorganisms can be
done only if aseptic technique is
practiced to prevent contamination by
other microorganisms.