Pharmaceutical Microbiology

Microscopic Examination of Microorganisms Practical Part Microscopic Examination of Microorganisms Experiments Identification of MOs Different Staining Techniques

Experimental Microbiology

Pour Plate Technique Objectives : To learn the pour-plate technique for using in other experiments such as streaking, sensitivity test and for MIC determination.

Materials : Procedure:- Tube of nutrient agar. Sterile Petri dish. Continue Materials : Tube of nutrient agar. Sterile Petri dish. Bunsen burner flame. Procedure:-

Place a tube of sterile nutrient agar in a boiling water bath Place a tube of sterile nutrient agar in a boiling water bath. Keep the water level at the halfway mark. (1)

Continue (2) 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.

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)

Continue (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.

Continue (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 : Procedure: (As previous) Six nutrient agar tubes. Continue Materials : Six nutrient agar tubes. Six sterile Petri dishes. Procedure: (As previous)

Source of Contamination No. of colonies of Source of Contamination Plate No. Actinomycetes Fungi Bacteria Air 1 Forced air 2 Cough 3 skin 4 Tap water 5 None 6

Continue 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.

Source of Contamination Results of Experiment: No. of colonies of Source of Contamination Plate No. Actinomycetes Fungi Bacteria Air 1 Forced air 2 Cough 3 skin 4 Tap water 5 None 6

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

Edge

Elevation

Colony description Isolate 1 Isolate 2 Isolate 3 Shape Color Size Edge (margin) Elevation Texture Surface Optical Character

Colony description Isolate 1 Isolate 2 Isolate 3 Shape Round , oval --------------------- Color Yellow Size Medium to large Edge (margin) Entire Elevation Convex Texture Smooth Surface Optical Character Opaque

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. Continue Materials : Tube of nutrient agar (10 ml). Sterile Petri dish. Wire inoculating loop. Tube containing mixed culture of two microorganisms. Permanent marker.

Continue Procedure: Invert the dish and by means of marker pen draw 5 groups, each with 3 parallel lines that intersect with each other.

Cool the agar to 50 ○C before pouring to avoid water condensation. Continue 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.

Continue 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 description Colony 1 Colony 2 Colony 3 Shape Color Size Edge (margin) Elevation Texture Surface Optical Character