Clinical Bacteriology - Laboratory - Isolation of Pure Culture of Organisms

Pure culture technique

This culture technique is done to identify the etiologic species/causative agent of a certain disease in a patient.

Pure culture technique

It will demonstrate how organisms grow as small distinct group in the medium. Small spots that occur in the medium present the growth of culture as distinct group.

Culture

act of cultivating or growing of organisms.

Pure culture

culture cultivating only a single species of organism.

Liquid

Broth

Liquid form of culture media

Agar

Solid form of culture media

Turbidity

What is the indication of the presence of bacteria in broth?

Colony

What is the indication of the presence of bacteria in agar?

Agar

It is the solidifying agent of solid culture media

It is not easily degraded by the microorganisms

Why do we use agar as our solidifying agent in solid culture media?

95°C or more

At what temperature does agar melt?

Below 50°C

At what temperature does agar solidify?

Colony

This is a bacterial population derived from a single bacterial cell (same genus and species)

Pure culture

What is the goal of isolating bacteria using culture media?

Pure culture

What culture is required for subsequent procedures used to identify and characterize bacteria?

Organism of interest

It is the organism we want to grow in the culture media

99.99%

When organisms from pure culture clone each other (asexual reproduction), how identical are the other organisms from each other?

Low

Pure culture multiplication rate (qualitative)

single

Pure cultures are composed of cells or microorganisms arising from a _x_ progenitor cells? (How many?)

Colony forming unit

CFU meaning

Colony forming unit

The progenitor from which a particular culture is derived

Aseptic technique

A technique performed under sterile conditions.

Streaking

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

Four quadrant streak plate method

Identify the setup

Qualitative

Four quadrant streak plate method: Qualitative or Quantitative?

To thin out inoculums to get separate colonies

What is the purpose of streaking the plate?

Four quadrant streak plate method

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

Sterilized wire loop

What equipment is used to streak the bacterial suspension onto the agar?

Subculture

This is done by streaking well-isolated colonies from the primary streak plate to a new plate.

Nutrient agar

Enrichment and non-selective solid culture media

3

How many loopfuls of the bacterial suspension are placed onto the agar?

Petri dish

a shallow dish used to culture bacteria

1/4

How much of the agar plate do you streak for the 1st quadrant?

2-3

When streaking the 2nd quadrant, how many times do you touch the 1st quadrant?

2-3

When streaking the 3rd quadrant, how many times do you touch the 2nd quadrant?

1

When streaking the 4th quadrant, how many times do you touch the 3rd quadrant?

24-48 hours at 37°C

Incubation time and temperature

Heat in-between quadrants

In the hospital setting, when we receive mixed culture, what is done during the streaking of agar plates in order to prevent contamination growing on the plates?

Biosafety cabinet

What equipment is required to streak the agar with the petri dish cover fully opened? (Not slightly opened)

30 degrees

How many degrees should you rotate your

Heavy confluent growth

1st quadrant growth

Less dense growth

2nd quadrant growth

Weak growth

3rd quadrant growth

Isolated single colonies

4th quadrant growth

4th quadrant

Which quadrant is pure culture obtained from?

1st quadrant

Which quadrant has tight streaks and heavy growth?

2nd and 3rd quadrants

Which quadrants have decreased density but may still contain mixed culture?

4th quadrant

Which quadrant is used for the identification of organisms?

Sterile petri dish

It is where the serial dilution and melted nutrient agar are poured together

Melted nutrient agar

What is poured together with the serial dilution for the serial dilution pour plate method?

Serial dilution pour plate method

Identify the setup

Quantitative

Serial dilution pour plate method: Qualitative or Quantitative?

Serial dilution pour plate method

In this method, the original sample is diluted several times, to reduce population sufficiently to obtain separate colonies.

Psychrophilic or cryophilic organisms

What organisms will not be able to grow using the serial dilution pour plate method?

Heat shock

What do the organisms in serial dilution pour plate method experience when mixed with melted nutrient agar?

45-50°C

At what temperature is melted nutrient agar maintained at?

1mL of inoculum, 9mL NSS

How is a 1:10 serial dilution made in a test tube?

1mL

What volume of bacterial suspension from the serial dilution is dispensed onto the serial dilution petri dish?

Quebec colony counter

What instrument is used to count colonies on the agar plate from the serial dilution plate?

1:10, 1:100, 1:1000

Which serial dilutions are "too numerous to count"?

1:10,000, 1:100,000, 1:1,000,000

Which serial dilutions produce single colonies on agar plates?

Pressure sensing device

Quebec colony counter principle

Felt-tipped pen

What equipment is used to count colonies on an agar plate on the Quebec colony counter?

Agar-side up

What is the orientation of the agar plate when it is incubated?

Moisture

What is the reason why an agar plate is incubated agar-side up?

Swirl on a flat surface

How is the 1mL bacterial suspension and melted nutrient agar mixed in the petri dish for preparation of the serial dilution plate?

Electronically

How do modern colony counters count colonies?

Felt-tipped pen

When counting colonies using the Quebec colony counter, what is used to mark the colony so that it is considered counted?

CFU/mL

Unit of measurement for colony count

Colony count x reciprocal of dilution

What formula (CFU/mL) is used for small number of colonies?

280,000 CFU/mL

Solve:

What is the colony count for a plate with 28 colonies on a plate of 1:10,000 dilution?

Colony count x reciprocal of dilution

What formula do you use for this situation?

Colony count: 15

Serial dilution of plate: 1:1,000,000

Average number of colonies in 5 squares x 62.5 x reciprocal of dilution sample

What formula (CFU/mL) is used for serial dilutions that are too numerous to count?

62.5

Which portion of the formula pertains to the area of the petri dish?

Average number of colonies in 5 squares x 62.5 x reciprocal of dilution sample

Colonial characteristics

It is the color, size, shape, surface appearance, changes in agar media, and odor of the colonies from the incubated agar plate.

Color

Which colonial characteristic refers to the pigmentation?

Size

Which colonial characteristic refers to the diameter?

Shape

Which colonial characteristic refers to the form, elevation, and margin?

Surface appearance

Which colonial characteristic refers to the appearance of the surface of the colony?

Changes in agar media

Which colonial characteristic refers to the changes resulting from bacterial growth?

Changes in agar media

Which colonial characteristic is not applicable to nutrient agar?

Odor

Which colonial characteristic refers to the smell?