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