Labs 2-4: Culture Techniques

Explain why the following steps are essential during subculturing:

a. Flaming the inoculating instrument prior to and after each inoculation

This is done to prevent contamination of the stock cultures

Explain why the following steps are essential during subculturing:
Holding the test tube caps in the hand as illustrated in Figure 2.1 on page 14

This is to prevent caps from touching surfaces that may contaminate it.

 Cooling the inoculating instrument prior to obtaining the inoculum

This is done because inserting a hot inoculating instrument will kill the cells.

Flaming the neck of the tubes immediately after uncapping and before recapping

Flaming the neck will kill of any organisms that might be present on neck of tube/inner surface of the culture

Describe the purposes of the subculturing procedure.

subculture is transferring microbes from one medium to another. the purpose of the of subculturing procedure is to ensure an aseptic transfer.

There is a lack of orange-red pigmentation in some of the growth on your agar slant labeled S. marcescens. Does this necessarily indicate the presence of a contaminant? Explain.

No, because bacterial genes may express themselves differently.

Upon observation of the nutrient agar slant culture, you strongly suspect that the culture is contaminated. Outline the method you would follow to ascertain whether your suspicion is justified.

I would sample the agar slant and transfer it to an agar plate.
Then i would incubate the plate to grow microbes.
I would then check to see if any unknown cultures grow.

Can you prepare a pure culture from a mixed-broth or a mixed-agar-slant

culture? Explain

Yes, you would just need to separate the mixed culture through dilution of the mixture or by doing a streak plate. This will give you individual colonies that you can then grow into a pure culture.

Observation of a streak-plate culture shows more growth in quadrant 4 than

in quadrant 3. Account for this observation.

This could be due to not properly sterilizing the inoculating loop or by accidentally spreading some of quad 1 into quad 4. This could also be due to a wider streak that made the microbe not diluted enough.

Why is a needle used to isolate individual colonies from a spread plate or

streak plate?

Less microbes can be picked up with a needle, limiting the possibility of getting 2 different microorganisms.

How can you determine if the colony that you chose to isolate is a pure

culture?

If the colonies are uniform in size, color, and morphology

culture characteristics.

observable traits of a microorganism when grown in a specific medium.

nutrient agar slants

1. Abundance of growth

2. pigmentation

3. optical characterists: opacity

4. form: appearence

5. consistency: dry, buttery, mucoid

nutrient agar plates

1. size

2. pigmenattion

3. form: circular, irregular, rhizoid

4. Margin: appearance of outer edge of the colony

5. Elevation

Nutrient Broth

1. uniform fine turbidity: finely dispersed growth throughout

2. flocculent: flakey aggregates dispersed throughout

3. pellicle: thick, padlike growth on surface

4. sediment: conc of growth at the btm of culture may be granular, flaky

nutrient gelatin

the solid medium may be liquefied due to gelatinase.
1. Crateriform: liquified surface area is saucer-shaped

2. Napiform: bulbous-shaped

3. Infundibuliform: Funnel-shaped

4. Saccate: Elongated, tubular

5. Stratiform: Complete liquefaction of the

   upper half of the medium

What is the function of the sterile loop or needle in transfer procedures?

• Loops: are used for picking up and transferring liquid cultures or spreading bacteria on agar plates. 

• Needles: are used for inoculating agar slants or deeps, or for picking up small, solid colonies. 

Describe the four-quadrant streak method.

1. Place a loopful of culture in Area 1. Flame the loop and cool it. Drag rapidly across the surface of Area 1.

2. Flame loop and turn plate 90 degrees. Touch the loop to corner of Area 1 and drag it across Area 2. the

3. Reflame the loop, cool it, and turn it again. Streak area 3 is the same as area 2.

4. Turn 90 degrees again and drag the culture from Area 3 to Area 4 using a wider streak. don’t let it touch any previous areas.

Why is it important to isolate single colonies?

allows study for indicual microbial studies, ensures purity of cultures, allows us to accurately identify organisms

What are three features used to describe colony morphology?

Size, shape , pigementation

How can broth cultures indicate motility?

by observing the spread of bacterial growth away from the initial inoculation point