Practical Part

Microscopic Examination

of Microorganisms Experiments

Identification of MOs

Different Staining

Techniques

Experime

ntal

Microbiol

ogy

Objectives :

To learn the pour-plate technique for

using in other experiments such as

streaking, sensitivity test and for MIC

determination.

Pour Plate Technique

Materials :

Tube of nutrient agar.

Sterile Petri dish.

Bunsen burner flame.

Procedure:-

• Place a tube of sterile nutrient agar in a boiling water

bath. Keep the water level at the halfway mark.

(1)

When the agar is liquefied, remove

the tube and allow it to cool to about

50°C.

Place an empty sterile Petri dish

before you, top side up.

(2)

• Pick up the tube of melted, cool agar, remove its closure.

•Flame the mouth of the open tube.(3)

• With your free hand, remove the top of the Petri dish and

quickly pour the agar into the dish.(4)

(5) Replace the Petri dish cover.

Gently rock the closed dish, or

rotate it in circular fashion on the

bench top, being careful not to allow

the still melted agar to wave up over

the edge of the bottom half or onto the

cover.

(6) Let the agar solidify without further

disturbance. When it is quite firm,

invert the plate.

Experiment 1

Distribution of Microorganisms in the

Environment

 

Objectives :

To demonstrate the wide distribution of

microorganisms in the air, on the skin, and

in the air droplet produced by coughing in

order to avoid sources of contamination.

Materials :

Six nutrient agar tubes.

Six sterile Petri dishes.

Procedure: (As previous)(As previous)

Plate

No.

Source of Contamin

ation

No. of colonies of Bacter

iaFungi Actinom

ycetes

1Air

2Forced air

3Cough

4skin

5Tap water

6None

Incubate all plates at room

temperature for 48 hr.

After incubation, examine the plates

for the presence of bacterial or fungal

colonies or actinomycetes and

describe each.

Results of Experiment:

Plate

No.

Source of Contamin

ation

No. of colonies of Bacter

iaFungi Actinom

ycetes

1Air

2Forced air

3Cough

4skin

5Tap water

6None

**Description of colonies from plate number (….)

Shape

Edge

Elevation

Colony descriptionIsolate 1Isolate 2Isolate 3

i. Shape

ii. Color

iii. Size

iv. Edge (margin)

v. Elevation

vi. Texture

vii. Surface

viii. Optical Character

Colony descriptionIsolate 1Isolate

2Isolate 3

i. Shape Round , oval------------

------------------------------

ii. ColorYellow

iii. SizeMedium to large

iv. Edge (margin)Entire

v. ElevationConvex

vi. TextureSmooth

vii. Surface

viii. Optical CharacterOpaque

Experiment 2

Techniques for Isolation of Pure

Cultures

 

Objectives :

To perform the spread plate and/or

streak plate inoculation procedure for

the separation of a mixed culture so

that discrete colonies can be isolated.

Materials :

Tube of nutrient agar (10 ml).

Sterile Petri dish.

Wire inoculating loop.

Tube containing mixed culture of

two microorganisms.

Permanent marker.

Procedure:

Invert the dish and by means of

marker pen draw 5 groups, each with

3 parallel lines that intersect with

each other.

Using pour plate technique,

aseptically pour the melted nutrient

agar into the plate and leave to

solidify.

N.B

Cool the agar to 50 ○C before pouring

to avoid water condensation.

After solidification, invert the plate to avoid

water condensation on the surface of agar.

• Gently Mix the Wasserman containing mixed culture.

• Flame the wire loop.

• Place a loopful of culture on the agar surface at

the beginning of group 1.

• Re-flame the loop between each group

Results of Experiment:

Colony descriptionColony 1Colony 2Colony 3

i. Shape

ii. Color

iii. Size

iv. Edge (margin)

v. Elevation

vi. Texture

vii. Surface

viii. Optical Character