Culture Media Preparation Review
( Modified from Supplemental Lecture by Stephen T. Abedon)
1.
2.
Culture
a.
A culture is the microorganisms that gro w in a culture medium.
b.
To culture means to gro w microorganisms in a culture medium.
Culture medium [pl. media]
a.
Culture media are solutions containing all of the nutrients and necessary physical gro wth
parameters necessary for microbial gro wth.
b.
Note that not all microorganisms can gro w in any given culture medium and, in fact, many
can't gro w in any known culture medium.
c.
In addition to chemical and physical characteristics, media can be distinguished
qualitatively as:
i.
solid vs. broth
ii.
non-synthetic vs. chemically defined
iii.
reducing
iv.
selective
v.
differential
3. Solid medium (agar)
a.
Solid medium is media containing agar or some other, mostly inert solidifying agent.
b.
Solid medium has physical structure (broth lacks structure) and this allows bacteria to
gro w in physically informative or useful ways (e.g., as colonies or in streaks).
c.
d.
Solid medium is usually used as:
i.
slants
ii.
stabs
iii.
petri dishes
See illustration below.
4. Colony
a. A colony is a pile or mass of a sufficiently large number of cells, gro wing on or in solid medium,
that they are visible to the naked eye.
5. Broth medium
a.
Broth medium is media lacking a solidifying matrix (see solid medium).
6. Non-synthetic [chemically undefined] medium
a.
Chemically undefined ingredient:
i.
Non-synthetic medium contains at least one component that is neither purified nor
completely characterized nor even completely consistent from batch to batch.
b.
ii.
Often these are partially digested proteins from various organismal sources.
iii.
Nutrient broth, for example, is derived from cultures of yeasts.
May be broth or solid.
7. Simple non-synthetic medium
a. A simple non-synthetic medium, operationally defined, is capable of meeting the nutrient
requirements of organisms requiring relatively fe w gro wth factors.
8. Complex non-synthetic medium
a.
Undefined with more gro wth factors:
i. A more complex non-synthetic medium, on the other hand, supplies more (additional
types of) gro wth factors.
ii. Complex non-synthetic medium are consequently able to support the gro wth of more
fastidious microorganisms than can simple non-synthetic medium.
9.Chemically defined [synthetic] medium
a.
A chemically defined medium is one prepared from purified ingredients and therefore
whose exact composition is known.
b.
Not trivial for fastideous:
i. To use a chemically defined medium you must kno w exactly what your microorganism's
gro wth requirements are.
ii. If this is not known it must be discovered through trial and error and therefore is not a
trivial process for fastideous microorganisms.
10. Preprepared medium
a.
Media of many types can be obtained premixed, in an often dehydrated and powdered
state. Such media are called preprepared media.
b.
Ready to use:
i. It is even possible to obtain sterilized media that is ready to use.
ii. Such media can be especially desirable if one lacks an autoclave, filtration devices, or the
desire to prepare even preprepared media from a can.
11. Enriched medium
a.
Added gro wth factors:
i. Enriched media is preprepared (or equivalent) media to which additional gro wth factors
have been added.
ii. These may be added individually or in complex mixtures.
iii.
Enriched medium therefore may be chemically defined or chemically undefined,
simple or complex.
b.
An example of enriched medium employing the latter would be blood agar which is made
from complex medium to which whole blood has been added.
12. Reducing medium
a. Oxygen scavenging media:
i. Reducing medium is employed for gro wing obligate anaerobes.
ii. Reducing medium particularly contains chemicals (reducing agents) that deplete molecular
oxygen.
13. Selective medium
a. Differential gro wth suppression:
i. Selective medium is designed to suppress the growth of some microorganisms while
allowing the gro wth of others (i.e., they select for certain microbes).
ii. Solid medium is employed with selective medium so that individual colonies may be
isolated.
b. Examples of selective media include:
i. mannitol salts agar (selects against non-skin flora)
ii. MacConkey agar (selects against gram-positives)
iii. eosin-methylene blue agar (selects against gram-positives)
iv. .phenylehyl alcohol agar (selects against gram-negatives)
14.
Differential medium
a. Differential appearance:
i. Differential media allow the gro wth of more than one microorganism of interest but with
morphologically distinguishable colonies.
ii. Note that almost any medium containing a specific substrate and well tailored indicator can
be used as a differential medium.
b. Examples of differential media include:
i. mannitol salts agar (mannitol fermentation = yellow)
ii. blood agar (various kinds of hemolysis)
iii. MacConkey agar (lactose fermentation = yellow)
iv. eosin-methylene blue agar (various kinds of differentiation)
15. Enrichment Culture
a. Techically enrichment culture differs from selective medium in not out and out suppressing
the gro wth of non-enriched microorganisms (e.g., no selective poisons are employed).
b. Enrichment culture is used to increase the relative concentration of certain microorganisms
in the culture prior to plating on solid selective medium.
c. Unlike selective media, enrichment culture is typically used as broth medium.
16. Pure culture technique
a. Clonal populations:
i. . Pure culture technique is a method of culturing microorganisms in which all of the
individuals in a culture have descended from a single individual.
ii.
This is done so as to:
1. inhibit evolutionary change within cultures
2. allow the characterization of types microorganisms without the confounding
presence of other, different types of microorganisms
b. Colony isolation:
i. iThe basis of pure culture technique is the isolation, in colonies, of individual cells,
and their descendants, from other colonies of individuals.
ii.
c.
This is usually done by culturing methods employing petri dishes such as:
1.
streaking
2.
pouring
3.
spreading
Isolation from the wild:
i.
When isolating microorganisms from complex mixtures it is always a good
idea to repeat the isolation procedure at least once (e.g., restreak an isolated
colony) to make sure that an isolated colony is truly derived from only a single cell
(i.e., closely overlapping colonies can be indistinguishable from colonies founded
from single cells).
ii.
Following their isolation from the wild, microorganisms may be
characterized by inoculation into differential medium to determine what type of
nutrients they require or can use, and what types of by-products they produce.
This aids in identification.
17.
Pouring a plate
a.
A pour plate is a method of melted agar inoculation followed by petri dish
incubation.
b.
Steps include:
i.
cultures are inoculated into melted agar that has been cooled to 45°C
ii.
the liquid medium is well mixed then poured into a petri dish (or vice versa)
iii.
colonies form within the agar matrix rather than on top as they do when
streaking a plate
c.
Pour plates are useful for quantifying microorganisms that gro w in solid medium.
d.
Because the "pour plate" embeds colonies in agar it can supply a sufficiently
oxygen deficient environment that it can allow the gro wth and quantification of
microaerophiles.
18.
Spreading a plate
a.
Quantification technique:
i.
Spreading a plate is an additional method of quantifying microorganisms
on solid medium.
ii.
Instead of embedding microorganisms into agar, as is done with the pour
plate method, liquid cultures are spread on the agar surface using a devise that
looks more or less like a hockey stick.
b.
An advantage of spreading a plate over the pour plate method is that cultures are
never exposed to 45°C+ melted agar temperatures.
19.
Preserving cultures
a.
b.
20.
Preserving cultures is important for:
i.
scientific reasons
ii.
identification
iii.
vaccine production
iv.
industrial use
v.
etc.
Methods of preserving cultures include:
i.
refrigeration
ii.
stabs
iii.
slants
iv.
lyophilization
v.
freezing
Refrigeration (a.k.a., 4°C)
a.
21.
Effective short term preservation:
i.
Refrigeration can be effective for short periods.
ii.
All of the following may be refrigerated:
1.
broth cultures
2.
stabs
3.
slants
4.
streaks
Stabbing
a.
Cultures are stabbed deeply into agar using a inoculating needle.
b.
The stabs are incubated until visible cultures form, then sealed and stored at room
or lower temperature.
c.
22.
See illustration.
Slant method
a.
Cultures may be streaked onto the surface of the solid medium in a slant tube.
b.
The slants are incubated until visible culture formation then sealed and stored at
room or lower temperature.
c.
23.
Lyophilization
a.
b.
24.
See illustration.
Freeze drying:
i.
Lyophilization is the freeze-drying of cultures.
ii.
Cultures are first frozen and then dried under high vacuum.
iii.
To revive cultures they are rehydrated by broth.
Lyophilization can be an effective long term method of storage.
Freezing
a.
Freezing with protection:
i.
Broth cultures are mixed with various ingredients (e.g., glycerol, DMSO) to
limit damage upon freezing and then frozen to temperatures ranging from -50°C to
-95°C.
ii.
b.
25.
To revive cultures they are thawed, pelletted, and resuspended into broth.
Freezing can be an effective long term method of storage.
Colony morphology
a.
Differentiating colonies:
i.
Colony morphology gives important clues as to the identity of their
constituent microorganisms.
ii.
Important classes of characteristics include:
1.
size
2.
type of margin
3.
colony elevation
4.
colony texture
26.
5.
light transmission
6.
colony pigmentation
Colony size
a.
Colony size is dependent not just on the type of organism but also on the gro wth
medium and the number of colonies present on a plate (that is, colonies tend to be smaller
w hen greater than ascertain amount are present) and on culture medium characteristics.
b.
Usually stabilizes after fe w days:
i.
Colony size usually stabilizes after a day or t wo of incubation.
ii.
Exceptions include:
1.
slo w gro wing microorganisms
2.
during gro wth under conditions that promote slow gro wth
iii.
With slow gro wth colonies may continue to experience gro wth past this
time, especially if an effort is made to prevent solid medium from drying out.
27.
28.
Type of margin
a.
Colonies can vary in the shape of their margins.
b.
See illustration below.
c.
llustration, variation in colony margins
Colony elevation
a.
Colonies can vary in their elevations both between microorganisms and gro wth
conditions, and within individual colonies themselves.
b.
See illustration below.
Illustration, variations in colony elevation
29.
Colony texture
a.
Surface appearance:
i.
Colonies can vary in their texture.
ii.
Possible textures include:
1.
shiny to dull
2.
smooth to wrinkled
3.
rough
4.
granular
5.
mucoid
b.
A shiny, smooth, and/or mucoid appearance tends to be associated with the
presence of capsular material.
30.
Colony light transmission
a.
The light transmission through colonies can range from:
i.
complete (transparent)
ii.
through intermediate (translucent)
iii.
through completely lacking (opaque)
31.
Colony pigmentation
a.
32.
Colonies can come in a rainbow of colors.
Petri dish
a.
Petri dishes are circular, vertical sided plates used to contain agar and with tops
for aseptic purposes.
33.
Loop
a.
A platinum wire formed into a loop is heated to an orange glow to sterilize it then
is used to transfer a culture from one physical location to another.
b.
34.
See illustration below.
Streaking
a.
Streaking is a method of applying cultures to solid medium:
i.
a sterile loop is cooled and brought into contact with a culture
ii.
the loop is then brought into contact with the surface of solid medium
w hereupon it is streaked (i.e., dragged) along the surface of the solid medium
iii.
b.
35.
colonies gro w along the points of the streak
See illustration below.
Streaking a plate
a.
Colony isolation:
i.
A petri dish is streaked in manner such that individual colonies may be
isolated.
ii.
See illustration below.
36.
Illustration streaking a plate
37.
Vocabulary - Define each of the following term s
a.
Broth medium
b.
Chemically defined medium
c.
Colony
d.
Culture
e.
Culture medium
f.
Differential medium
g.
Enriched medium
h.
Isolation from the wild
i.
Non-synthetic medium
j.
Pouring a plate
k.
Preserving cultures
l.
Pure culture technique
m.
Reducing medium
o
n.
Selective medium
o.
Solid medium
p.
Solid medium, illustration
q.
Spreading a plate
r.
Streaking a plate
s.
Streaking a plate, illustration
t.
Variation in colony elevation, illustration
u.
Variation in colony margins, illustration
Practice questions
a.
To purify a microorganism, which would you very likely utilize (circle only one
correct ans wer)?
i.
solid medium
ii.
broth medium
iii.
non-synthetic medium
iv.
chemically defined medium
v.
all of the above
vi.
none of the above
b.
You have a microorganism you know is both fastidious and which normally lives
in human lungs. Which medium might this organism have the highest probability of
gro wing on? (choose best ans wer)
i.
simple non-synthetic media
ii.
complex non-synthetic media enriched with fresh, whole blood
iii.
chemically defined, minimal media
iv.
complex non-synthetic media
v.
all of the above
vi.
none of the above
c.
Design an enrichment medium and a procedure for gro wing an endospore-forming
bacterium that fixes nitrogen and uses cellulose for its carbon source (from Tortora et al.,
1995, p. 166).
d.
A medium which lacks physical structure would be called _____________?
e.
You figure that if you could come up with a bacteria that decomposed PCBs you'd
make zillions (PCBs---a.k.a., polychlorinated biphenyls---are a heterogeneous kind of
toxic waste that are at least potential carbon and energy sources). In setting forth on this
task you note that PCB spills often occur into environments both lacking in sufficient
oxygen supplies and having relatively low temperature (e.g., the muck at the bottom of
bodies of water), though it would be handy if the organism was also capable of gro wth
in the presence of atmospheric oxygen and at 25°C. You figure that for handling reasons
you would also prefer that this organism be capable of producing endospores. How
w ould you isolate bacteria that could decompose environmental PCBs?
f.
List two methods of efficient long term bacterial storage.
g.
If you were interested in isolating a wild microorganism in pure culture, one which
you suspect makes up approximately half of the cells in your crude mixture, name a
technique (other than the use of selective media or enrichment culture) that you would
have to employ in order to accomplish this isolation, and tell me a key aspect of ho w you
w ould have to use it in order to assure the preparation of a truly pure culture.
h.
Fill in the blanks with (a) reducing medium, (b) differential medium, (c) selective
medium, (d) enriched medium, or (e) enrichment culture.
i.
Agar containing 8.5% CaCl2 to increase osmolarity of medium:
___________.
ii.
Broth containing molecular oxygen (O2)-scavenging chemical:
__________.
iii.
Agar containing factors which inhibit gram-positive bacteria gro wth, such
as bile salts and crystal violet: __________.
iv.
i.
Agar which turns blue when galactose is fermented: __________.
Which method is least able to maintain cultures for the long term (years):
i.
stabbing
ii.
slant culture in refrigerator
iii.
lyophilization
iv.
storage at -85°C
v.
storage at -70°C
vi.
stab culture in refrigerator
j.
What does the noun "isolate" mean?
k.
A __________ medium is one that aids the gro wth of obligate anaerobes.
l.
A __________ medium is designed to suppress the gro wth of some
microorganisms while allowing the gro wth of others.
i.
reducing
ii.
selective
iii.
differential
iv.
enriched
v.
complex
vi.
chemically defined
References
Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper
Saddle River, New Jersey. pp. 144-148.
Cappuccino, J.G., Sherman, N. (1996). Microbiology. A Laboratory Manual. Fourth Edition. The
Benjamin/Cummings Publishing Co., Inc. Menlo Park, California. pp. 13-16, 21-23, 89-90.
Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction. Fifth Edition. The
Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 147-154, 158-166.
Ans wers
a.
i, solid medium
b.
ii, media enriched with fresh, whole blood. The fact that it's fastidious and lives in/on you
suggests that it might require gro wth factors normally present in the human body. A good
sampling of those gro wth factors often can be found in whole blood.
c.
No nitrogen except from the atmosphere and no carbon source except from cellulose.
Boil the mixture at some point, preferably before inoculating, to kill all vegetative cells. Then you
would isolate the surviving endospores perhaps on selective media (i.e., no nitrogen with
cellulose as its sole carbon source).
d.
broth medium
e.
Start with the muck at the bottom of a lake (or a soil sample). Boil sample to kill vegetative
cells. Use selective media containing only PCBs as both the carbon and energy source. Incubate
at low temperatures and under anaerobic conditions. Plate differentially on PCB-containing plates
(sole carbon and energy source) at 25°C, exposed to the air.
f.
Freezing, lyophilization, and to a lesser extent stab culture.
g.
Colony isolation by streak or pour plate and repeated isolation of individual colonies in
order to assure purity.
h.
selective medium, reducing medium, selective medium, differential medium.
i.
ii, slant culture in refrigerator.
j.
An isolate is a (preferentially pure) culture of microorganisms which was derived from a
heterogeneous wild population.
k.
Reducing ii, selective