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Culturing Microbes

Eri Dian
Bacteria have to be grown (cultured) for
them to be identified.
By appropriate procedures they have to be
grown separately (isolated) on culture
media and obtained as pure for study.
History
The original media used by Louis Pasteur
urine or meat broth
Liquid medium diffuse growth
Solid medium discrete colonies.

Culturing Microbes

The Five Is
Innoculation: Producing a pure culture
Isolation: Colony on media, one kind of microbe,
pure culture
Incubation: growing microbes under proper
conditions
Inspection: Observation of characteristics (data)
Identification: use of data, correaltion, to ID
organism to exact species
Culturing Microbes
The Five Is
Innoculation: Producing a pure culture
Introduce bacteria into a growth medium using aseptic technique to
prevent contamination. Tools: Bunsen burner, loop. Needle, etc.
Microbiology
Innoculation: Producing a pure culture
Introduce bacteria into a growth medium using aseptic technique to
prevent contamination. Tools: Bunsen burner, loop. Needle, etc.
Microbiology
Isolation: Colony on media, one kind of
microbe, pure culture: isolation on general
and special differential media

General growth media: Nutrien Agar (NA),
Triple sugar Agar (TSA)
Differential: Mac, EMB, SS
These have dyes, salts, inhibiting agents :
see differences on plates

Microbiology
Isolation: Colony on media, one kind of
microbe, pure culture

Microbiology
Isolation: Colony on media, one kind of
microbe, pure culture Streak Plates

Microbiology
Isolation: Colony on media, one kind of
microbe, pure culture. Many colonies? Use
a needle, pick one, and redo streak plate

Microbiology
Differential: Mac, EMB, SS
These have dyes, salts, inhibiting
agents : see differences on plates

Microbiology
Blood agar : rich with nutrients, can see a
difference, thus differential; much more
later
Microbiology
Incubation: Allow organisms to grow under the optimal
conditions
Temperature, with or without oxygen etc
Microbiology
Incubation: Allow organisms to grow under the optimal conditions
Temperature, with or without oxygen etc
Candle jar reduces oxygen

Microbiology
Inspection: Observation, description
Colony Morphology, Microscopic examination (grams
stain)
Systematic recording of DATA
Microbiology
Microscopic study: Gram + bacilli, Gram -
bacilli
Microbiology
Microscopic study: Acid fast, and capsule
Microbiology
Identification: Correlating data from all observations to
ID organism to species
Resources: flow charts, Bergeys manual etc.
Ex. Gram bacilli, ferments lactose, green sheen on
EMB: E.coli
Microbiology
Identification: Correlating data from all observations to
ID organism to species
Gram + cocci, grape like clusters, golden yellow colonies, catalase
+, coagulase +, resistant to Methicillin (MRSA)
Staphylococcus aureus
Type and Selection Of Culture
Media
Basic
Enriched
Selective
Exclusive
Differential
Transport
Basic media
Simple media
To support the growth of m.o that do not
have special nutrional requirements
Nutrien agar or nutrien broth
Often used to maintain stock culture of
control strain
Enriched Media

Media that are enriched wth whole blood,lyzed
blood,serum,extra peptone,or vitamin
To support the growth of pathogens that require
additional nutriens or growth stimulants
Haemophilus influenzae, Streptococcus sp
The term enrichment is used to describe a fluid
medium that increases the number of pathogens
by containing enrichments,and or subtances that
discourage the multiplication of unwanted
bacteria
Blood agar,chocholate agar
Selective Media
Media which contain substances that prevent or
slow down the growth of m.o other than the
pathogens for which the media are intended.
Example:
SS (Salmonella shigella) agar selects for
Salmonellae and Shigellae and inhibit the
growth of many faecal commensals
Differential (Indicator)Media
These are media to which indicators,dyes,or
other substances are added to differentiate m.o
Ex:TCBS (thiosulfate cytrate bile sucrose) agar
contains the bromotymol blue indicator which
differentiate sucrose fermenting from non-
sucrose fermenting Vibrio-species
Endo agar that differentiate lactosa fermenter
(Ex. E.coli) and non lactose fermenter
(Salmonella, Shigella)
Transport Media
These are mostly semisolid media
That contain ingridients to prevent the
overgrowth of commensals
And ensure the survival of aerobic and
anaerobic pathogens when specimen
cannot be cultured soon after collection
Ex: Carry Blair, Amies, Stuart,Charcoal
Solid,Semisolid And Fluid
Culture Media
Culture media can be used in three forms:
Solid,semisolid, Fluid

Solid culture media
When grown on solid media,m.o multiply
to form visible colonies
When viewed from above, colonies may
appear round,irregular,branching
When viewed from side, colonies may
appear flat or raised
When touched wth a wire loop, some
colonies are soft and emulsified such
S.aureus
Fluid media
Are used as enrichment
media,biochemichal test
Semisolid media
Is prepared by adding a small amount of
agar to a fluid medium
Used mainly as transport media and for
motility testing

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