Exercise 5 - Isolation of Pure Culture of Organisms

PURE CULTURE TECHNIQUE

to identify the etiologic species/causative agent of a certain disease in a patient

CULTURE

act of cultivating or growing of organisms

PURE CULTURE

cultivating only a single species of organism

• LIQUID

• SOLID

Different types of culture media:

PRESENCE OF TURBIDITY

Indication of bacteria present in LIQUID culture media:

SOLID

it has a hardening or solidifying agent – AGAR

AGAR

excellent hardening agent, because most microorganisms cannot degrade it, therefore microorganisms can grow through agar

COLONIES

Small spots that occur in the medium are known as

COLONY

bacterial population derived from a single cell

OBTAIN PURE CULTURE

GOAL:

SINGLE PROGENITOR CELL

Cultures composed of cells/microorganisms arising from a

PROGENITOR

is termed a CFU (Colony Forming Units)

COLONY FORMING UNITS

Progenitor is termed a

• FOUR QUADRANT STREAK PLATE METHOD

• SIMPLE DILUTION POUR PLATE METHOD

Common Isolation Techniques:

FOUR QUADRANT STREAK PLATE METHOD

QUALITATIVE METHOD

To thin out inoculums to get separate colonies

Purpose of streaking the plate in four quadrant streak plate method:

STREAKING

the process of spreading the microbial culture with an inoculating needle on the surface of the media

PRIMARY STREAK PLATE TO A NEW PLATE

Subculture can be done by streaking well isolated colonies from the

PURE CULTURE

The one that is streaked in the new plate is the

1ST QUADRANT

tight streak, heavy growth

QUADRANT 1

no pure colony (may contain mixed culture)

2ND – 3RD QUADRANTS

density decreases

2ND – 3RD QUADRANTS

may still contain mixed culture

QUADRANT 4

consists of pure culture

4TH QUADRANT

used for the identification of organisms

SERIAL DILUTION POUR PLATE METHOD

QUANTITATIVE METHOD

SERIAL DILUTION POUR PLATE METHOD

The bacterial culture and liquid agar medium are mixed together

MELTED NUTRIENT AGAR

After mixing the medium, the medium containing the culture poured into sterilized petri dishes (petri plates), allowed solidifying and then incubate.

o Solidify because we are using

• Microorganism trapped beneath the surface of medium

• Tedious and time-consuming method, microbes are subjected to heat shock

• Psychrophilic or cryophilic organisms will not be able to grow

DISADVANTAGES OF POUR PLATE METHOD:

HEAT SHOCK

Tedious and time-consuming method, microbes are subjected to

• 1 ML OF INOCULUM

• 9 ML OF BROTH

1:10 comprises of

QUEBEC COLONY COUNTER

For pour plate method, the number of colonies are counted using the

• 1:10

• 1:100

• 1:1,000

TNC – Too Numerous to Count

• 1:10,000

• 1:100,000

Can see single colonies

QUEBEC COLONY COUNTER

It is an instrument used to count colonies of bacteria or other microorganisms that grows on an agar plate

SUBCULTURE

Can be done by streaking well isolated colonies from the primary streak plate to a new plate

• CHANGES IN AGAR MEDIA

• ODOR

• PIGMENTATION

• SHAPE

• SIZE

• SURFACE APPEARANCE

COLONY CHARACTERISTICS:

SINGLE BACTERIAL CELL

Colony is derived from:

NUTRIENT AGAR

Medium to use for FOUR QUADRANT STREAK PLATE METHOD

MELTED NUTRIENT AGAR

Medium to use for SIMPLE DILUTION POUR PLATE METHOD

62.5

Area of Petri Dish

PURE CULTURE

Required for subsequent procedures due to ID and characterize identify bacteria

Heavy confluent growth

QUADRANT 1

Weak growth

QUADRANT 2

Less dense growth

QUADRANT 3

Isolated single colonies

QUADRANT 4

EARLY COLONY COUNTER

Plates are placed under a magnifying, marking and counting colonies using a felt-tipped pen