Name: Sappayani, Nurhaifa S.

Section: Bsn II-E Activity 4 Gram Staining

Objectives: At the end of this experiment, the students should be able to: Demonstrate and perform the Grams staining procedure. Enumerate and understand the principles of this procedure and; Differentiate between Gram-negative and Gram-positive microorganism

Materials: Prepared fixed slude of Stapylococcus aureus and Escherichia colli Grams Reagents Crystal violet Grams iodine Ethanol Safranin Bunsen burner and alcohol lamp Staining rack Distilled water Wash bottle Lens paper/tissue paper Compound microscope Immersion oil

Procedure: Lay the prepared fixed bacterial smear on the staining rack. Flood the slide with crystal violet for 60 seconds. Wash with tap water for no more than 5 seconds. Flood the slide with grams iodine for another 60 seconds. Rinse the slide with water for roughly 5 seconds. Decolorize by flooding with 70% ethyl alcohol for 30 second until the blue or purple color will be washed out. Rinse under running water. Counterstain with dilute safranin for 30-60 seconds. Wash hunder running water for 5 seconds. Blot dry with tissue paper. Do not rub the smear. Examine under microscope. Observe and record the results. Using colored penals, sketch the organisms and differentiate the gram positive cells with gram negative cells. Take note of the shape and arrangement and record accordingly.

Interpretation: Gram positive cells with incorporated little or no counterstain and will remain blue-violet in appearance, Gram negative bacteria, however take on a pink color and are easily distinguishable from those that are the Gram positive.

Name: Sappayani, Nur-haifa S. Section: Bsn II-E

Activity 5 Culture Media Objectives: At the end of the experiment the students should be able to: Evaluate the different types of culture media; Identify the different classification of culture media Explain the principles of employed in the preparation of commonly used culture media. Materials: Dehydrated media Distilled water Defilarinated blood Autoclave Hotplate with stirren Weighing scale Petri dishes Erlenmeyer flasks Cotton Aluminum foil Autoclave incinerator tape

Procedure: bottle. A complete set of instruction for the preparation of culture media is given on the label of each

Use warm distilled water to hasten the solution of the medium. Rinse all glassware with distilled water and make sure that all vessels are clean. Prepare the medium in a flask about twice the final volume of the medium to allow adequate mixing follow the instructions given of the label of each product. Open the culture medium container in a cool dry place. Avoid inhaling the provider and prolonged skin contact weighs the provider quickly and accurately. Reclose the container as soon as possible. Pour half the required volume of distilled water in a flask, and then pour the weighed quantity of medium and mix briskly for a few minutes. Pour the rest of the distilled water down the slide of the vessels to wash any adherent media medium back into solution. Media containing agar should be healed to dissolve the agar before autoclaving. Bring the medium to boil the hot plate, stirring constantly without burning. The resulting solution should be dear lightly yellow with little or no undissolved materials. Allow the solution to cool cover the mouth of the flask lightly with a cotton plug wrapped with aluminum foil and finally sealed with an autoclave indicator tape. Sterilize the solution in the autoclave at 121 degree Celsius for 20 minutes under the supervision of your laboratory instructor. Liquid media which are sterilized in their final container should be cooled down to room temperature as rapidly as possible screw caps should then be lightened container of agar media which have been sterilized should be placed in a 50 degree Celsius water bath and the medium of dispensed as soon as it reaches this temperature or within a maximum of 3 hours in the bath. Blood used for the preparation of blood agar should be as fresh as possible. Mix the defibrinated blood(5%0 with the sterile molten agar base which has been cooled to 40-45 degree Celsius for chocolate agar, add the defibrinated blood at a higher of about 75-80 degree Celsius. Aseptically dispense the culture media in to petri dishes. Pour about 20ml/petri dish. One liter of medium will fill 50 standard petri dishes then allow the solution in the petri dishes to cool down further until it suddifies. The recommended shelf life of prepared culture media varies considerably screw capped battled of nutrients broth and agar can be stored for 6 months at low ambient temperature(12-16 degree Celsius). Agar plates should be stored at 2-8 degree Celsius in sealed containers to avoid loss of moisture. It is important to store all media away from light. Do not freeze.

Activity 6 (Acid Fast staining) cold method Objectives: To be able to identify acid fast bacilli and understand the principle of AFB staining. Procedure: Make a bacterial smear by spreading a loopful of sputum on a clean glass slide uniformly in oval-shape by smearing repeatedly in coil-like pattern approximately 1x2cm or 2x3cm in size. Air dry: fix over flame. Stain using the ziehl neilson rg. Flood smear with carbol fuchsin(Primary dye) for 10 mins. Wash in runnig water. Decolorized with acid alcohol until no more stain is visible. Counter stain with methylene blue. Rinse in water: air dry. Interpretation: AFB stain red or pink non-acid fast stain blue.

Observation/ Results: Draw what you observe under the microscope. Give example of acid fast bacilli other than M.tuberculosis. Give some common sites (pathogenesis of other than TB of the lung tuberculosis.

Activity 7 (Specimen Collection) Objectives: *To be able to know how to collect sputum for AFB stain and for Culture/sense.

Materials: Sputum specimen Sterile container BAP/ chocolate plate Alcohol lamp Glass slides

Procedure: How to collect: instruct the patient for AFB smear. -

How to collect sputum for C/S.

Observation/results: Manner of reporting the AFB (Report in scale)