L5: THE ISOLATION OF PURE CULTURE ORGANISMS

What is bacterial cultivation?

Growing microorganisms in culture.

Liquid form

(Broth Dilution) → Turbidity

Solid form

(Agar Dilution) → Colonies

How do you check for positive results in bacterial culture? (Broth Dilution)

Turbidity after 24-48 hours of incubation.

How do you check for positive results in bacterial culture? (Agar Dilution)

Presence of colonies after 24-48 hours of incubation.

What medium/material is used for broth dilution?

Nutrient broth

What medium/material is used for agar dilution?

Melted Nutrient Agar

What is the purpose of obtaining a pure culture?

To identify and characterize bacteria.

What is the goal of pure culture isolation?

To obtain a single species of bacteria for study.

What percentage of genetic similarity do organisms in a pure culture share?

99.999% genetically identical to the parent strain.

What is the importance of a pure culture in microbiology?

• Ensures only the desired microorganism is being grown.

• Allows accurate identification and diagnosis.

• Ensures consistency in experimental results.

How is a pure culture usually obtained?

By isolating bacteria from a mixed culture using sterile growth media.

What are colonies in a solid culture medium?

Macroscopic clumps of bacteria formed by repeated cell division.

What is a CFU (Colony Forming Unit)?

A single progenitor cell that gives rise to a colony.

Why is aseptic technique important in bacterial isolation?

Prevents contamination of sterile substances or objects.

What are the common isolation techniques?

• Streak Plate Method

• Spread Plate Method

• Pour Plate Method

What are the two types of isolation methods?

• Quantitative: Serial Dilution

• Qualitative: Four Quadrant Streak Plate

Area 1

Heavy confluent growth

Area 2

Less dense growth

Area 3

Weak growth

Area 4

Isolated single colonies

What is the principle behind streaking?

A dilution gradient is established on the agar surface as cells are deposited.

Why is normal saline solution used in bacterial suspension?

It provides a normal environment for the bacteria.

What is the purpose of streaking?

To thin out inoculums to get separate colonies.

Why is the agar plate incubated upside down?

To prevent condensation moisture from merging separate colonies.

What is subculture?

The transfer of microorganisms from an existing culture to a fresh medium.

Why is subculture performed?

To maintain viability and purity of organisms in the lab.

Where should colonies for subculture be taken from in a streak plate?

The 4th quadrant, as it contains pure culture.

What is the principle behind the Serial Dilution Pour Plate Method?

Bacterial culture is diluted and mixed with liquid agar for colony isolation.

What is the best dilution for obtaining a pure culture?

1:1,000,000 dilution.

What is a 10-fold serial dilution?

Each step reduces the concentration by a factor of 10.

What is the chemical composition of Nutrient Agar?

5g Peptone, 3g Beef Extract, 8g Sodium Chloride, 15g Agar, 1L Water.

What is a disadvantage of the pour plate method?

Microorganisms can be trapped beneath the agar surface, making counting difficult.

What is the standard temperature for cooling nutrient agar before pouring?

45-50°C.

Why is the petri dish swirled after pouring the agar?

To distribute the medium evenly.

How long is the plate incubated in the Serial Dilution Pour Plate Method?

24-48 hours at 37°C.

What is a Quebec Colony Counter?

An instrument used to count bacterial colonies on an agar plate.

What is the principle behind a Quebec Colony Counter?

It uses a pressure-sensing device to count colonies.

How were early colony counters used?

Colonies were marked and counted manually using a felt-tipped pen.

How do modern colony counters work?

They count colonies electronically when touched with a pen.

How do you calculate the number of CFU/mL?

Colony count × dilution factor.

What should you do if the colony count is too numerous to count?

Count colonies in 5 squares, multiply by 62.5, then multiply by the dilution factor.

Color

Pigmentation of the colony.

Size

Diameter of the colony.

Shape/Form

Basic shape of the colony.

Edge/Margin

Shape of the colony’s edges.

Surface

Appearance of the colony surface.

Elevation

Side view of the colony.

What serial dilution is commonly used?

10-fold dilution.

How should you answer when identifying the isolation method?

Either Four-Quadrant Streak Plate Method or Serial Dilution Pour Plate Method.

How do you determine the dilution in a labeled test tube?

Identify the dilution pattern (e.g., 1:10, 1:100, etc.).

What is the function of an alcohol lamp?

Used for sterilization.