Exercise V- The Isolation of Pure Culture of Organisms

Pure culture technique

It is done 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.

AGAR

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

COLONY

bacterial population derived from a single cell.

aseptic technique

Apply —- to prevent contamination of sterile substances or objects.

• Four Quadrant Streak Plate Method

• Serial Dilution Pour Plate method

Common isolation techniques:

FOUR QUADRANT STREAK PLATE METHOD

• Qualitative method

• By streaking, a dilution gradient is established on the surface of the plate as cells are deposited on the agar surface.

Streaking

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

SUBCULTURE

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

• The one that is streaked in the new plate is the pure culture of the organism

nutrient agar

medium used in four quadrant pour plate method

24-48 hours

incubation for the four quadrant streak plate method

heavy confluent growth

1st quadrant growth

Less dense growth

2nd quadrant growth

weak growth

3rd quadrant growth

isolated single colonies

4th quadrant growth

SERIAL DILUTION POUR PLATE METHOD

• Quantitative method

• Original sample is diluted several times, to reduce microbial population sufficiently to obtain separate colonies (isolate pure culture).

• The bacterial culture and liquid agar medium are mixed together.

melted nutrient agar

medium used in serial dilution pour plate method

• Microorganism trapped beneath the surface of medium hence surface as well as subsurface colonies are developed which makes it difficult to count bacterial colonies.

• Tedious and time-consuming method, microbes are subjected to heat shock because liquid medium maintained at 45-50°C.

• Psychrophilic or cryophilic organisms will not be able to grow

disadvantages of serial dilution pour plate method

37°C for 24 to 48 hours

incubation for serial dilution pour plate method

Quebec Colony Counter.

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

• Pressure Sensor Device. By touching it with a felt-tipped pen, there is a monitor on the Quebec colony counter which would count the colonies. • Marker is used to help remind that colony has been counted. (no repetition of counting)

CFU/mL = colony count x reciprocal of dilution sample

SMALL NUMBER OF COLONIES formula

CFU/mL = average # of colonies in 5 squares x 62.5 x reciprocal of dilution sample

TOO NUMEROUS TO COUNT (ESTIMATE) formula

Pressure-sensing device

What is the principle behind Quebec Colony Countrr

a dilution gradient is established on the surface of the plate as cells are deposited on the agar surface

Principle behind Four Quadrant Streak Plate Method