PROCESSING SPUTUM SAMPLE INTRODUCTION The material which is coughed up from the lungs and then spat out

or expectorated is called sputum or sometimes referred to as phlegm. A sputum culture is done to identify the microorganism causing lower respiratory tract infections such as pneumonia and tuberculosis. A fever with a chronic cough , along with blood or pus-like material in the sputum, is usually an indication for undertaking a sputum culture. The sputum should be collected in a sterile container, preferably early in the morning before eating or drinking anything. The mouth should be rinsed with water to rinse out bacteria from the mouth and dilute the saliva which may contaminate the specimen. With a forceful cough, the sputum should be spat out into the sterile container immediately, avoiding prolonged collection in the mouth cavity. Three consecutive samples may have to be collected if testing for tuberculosis. A special stain called the acid-fast stain may be done in the laboratory to identify the tuberculous bacilli. Different types of microorganisms may be identified using gram stain. A fungal culture may be done if a fungal infection is suspected and a viral culture is done to detect viral infection such as pneumonia. Due to the prevalence of bacterial respiratory tract infections, most sputum samples are first tested for bacteria. Bacterial Culture The initial report indicating the presence of any bacteria may be available on the same day. The final report will take one to three days and this will include identification of the specific type and quantity of bacteria as well as the antibiotics most effective against it. Culture for tuberculosis may take two to four weeks. Fungal Culture Reports may take several weeks. Viral Culture

It may take several days to several weeks to get the report depending on the type of virus present. OBJECTIVES 1) To identify the existence of Mycobacterium Tuberculosis in sputum sample cone from patient. 2) To study the procedures of identifying the suspected bacteria in sputum sample. MATERIALS Sputum sample Sterile collection swab Bunsen burner Slides Preparation for Gram Stain (crystal violet, iodine, alcohol and safranin) Preparation for Acid Fast Stain (carbolfuschin, acid-alcohol, methylene blue) Preparation for catalase, coagulase, and oxidase test Preparation for antibiotic-sensitivitity test Blood agar, Mc Conkey Agar, SDA agar Light Microscope

PROCEDURES

1) Smear of organism that to be stained were prepared. 2) Heats fix the smear. 3) The slide placed on wire gauze on a ring stand then was put with carbolfuhsin. 4) Heat the slides with a hand-held bunsen burner until steam can be seen rising from the surface. Alternately remove the burner and reheat the slide to maintain steaming for 3-5 minutes. As the paper begins to dry during the staining process add a drop or two of carbolfuschin to keep the slide moist. Adding too much stain will cool the slide (and drip on the bench). Overheating the slide or letting it dry will distort the cells. Under heating the slide will fail to stain acid-fast cells. 5) At the end of staining remove the paper with tweezers and wash the slide thoroughly. 6) Drain the slide. Decolorize with acid-alcohol for 30 seconds. 7) Rinse, drain, and counterstain with methylene blue for 45 seconds. 8) Rinse, blot, and examine. First observe each organism on its separate smear. Then examine the mixed smear. 9) Acid-fast organisms will appear red and non-acid-fast organisms will be blue. 10) Procedure proceeds on grams stain. 11) Then, identify it under microscopes. Results were recorded. 12) The bacteria streaked on Blood Agar, SDA Agar using germ tube, and Mc Conkey Agar. Then, they were incubated for 24 hours. Same goes to antibioticsensitivity test. 13) On the next day, if the results were gram positive, then coagulase, catalase and oxidase test were run otherwise, run with TUM IMVIC test.

RESULTS

1) The gross appearance of sputum showing sticky, cloudy figure. 2) After gram stain were done, the results showing the existence of gram positive cocci and fungi. Meanwhile, for the acid fast stain, Mycobacterium Tuberculosis were found.

1000x magnification gram stain stain

1000x magnification acid fast

3) On the next day, there were growths with alpha-hemolytic on blood agar, no growth in McConkey Agar, and the germ tube results negative. 4) Then, when examine under microscope, gram positive in chain were found then undergoes gram positive test.

1000x magnification gram stain

5) Catalase test presenting positive result, coagulase test presenting positive result, and oxidase test presenting negative results.

6) The antibiotic-sensitivity test showing: - Fucidic acid = 10mm - Gentamycin = 8mm - Erythromycin = resistant - Penicillin = resistant 7) The examination did not proceed by using Lowenstein-Jensen medium to identify MTB bacteria.

DISCUSSION

MTB bacteria did not grow in blood agar as well as in Mc Conkey agar but it grow on Lowenstein-Jensen medium. The procedure did not proceed to that step because it needs 3 weeks to see the result.Lowenstein-Jensen (LJ) medium is a growth medium for culture Mycobacterium notably Mycobacterium Tuberculosis. When grown on LJ medium, M. tuberculosis appears as brown, granular colonies (sometimes called "buff, rough and tough"). The media must be incubated for a significant length of time, usually four weeks, due to the slow doubling time of M. tuberculosis compared with other bacteria (15-20 hours).Gram positive shown staphylococci and might be Staphylococcus Aureus due to the results of biochemical test. Acid fast stain used to to differentiate between acid-fast and non acid-fast bacteria. Some bacteria contain a waxy lipid, mycolic acid, in the cell wall. This lipid makes the cells more durable and is commonly associated with pathogens. Acid fast cell walls are so durable that the stain (carbolfuschin) must be driven into the cells with heat. The cells are then decolorized with acid-alcohol, all other cells will decolorize with this strong solvent, but acid fast bacteria will not. Other cells are then counterstained with methylene blue to give it color.These bacteria resistant with erythromycin and penicillin but more sensitive with fusidic acid. One important use of fusidic acid clinically is its activity against methicillinresistant Staphylococcus aureus. Many strains of MRSA remain sensitive to fusidic acid, but because there is a low genetic barrier to resistance (a single point mutation is all that is required), fusidic acid must never be used on its own to treat serious MRSA infection and should be combined with another antimicrobial such as rifampicin.

CONCLUSION The MTB bacteria did not identified due to the length of time to incubate it with LJ medium, approximately 3 to 4 weeks. The Staphylococcus Aureus were recognized in this sample.