Nursyazmin Binti khairuddin (12273208031) Pathogenic Microbiology (HLB 22303)

LAB REPORT 7(b): SPUTUM SAMPLE 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.

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.

Nursyazmin Binti khairuddin (12273208031) Pathogenic Microbiology (HLB 22303)

Viral Culture It may take several days to several weeks to get the report depending on the type of virus present. Materials: 1. Sputum sample 2. Blood agar 3. McConkey Agar 4. SDA agar 5. Sterile collection swab 6. Bunsen burner 7. Slides 8. Light Microscope 9. Preparation for Gram Stain - (crystal violet, iodine, alcohol and safranin) 10. Preparation for Acid Fast Stain - (carbolfuschin, methylene blue) 11. Preparation for catalase, coagulase, and oxidase test 12. Preparation for antibiotic-sensitivitity test. acid-alcohol,

Method: 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.

Nursyazmin Binti khairuddin (12273208031) Pathogenic Microbiology (HLB 22303)

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 antibiotic-sensitivity 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. Result: Macroscopic view of sample: Sticky and cloudy figure

Microscopic view: Sample:

Gram stain (G+ cocci and fungi)

Acid fast stain (Mycobacterium Tuberculosis)

Colony:

Nursyazmin Binti khairuddin (12273208031) Pathogenic Microbiology (HLB 22303)

Gram positive cocci in chain (Blood agar)

Culture characteristics: Blood agar = alpha-hemolytic McConkey Agar = RI then no growth SDA agar = RI Germ tube (SDA agar) = negative

Biochemical test: Catalase test = positive Coagulase test = positive Oxidase test = negative

Sensitivity testing: Fucidic acid (FD 10) = 10mm Gentamycin (Gen.) = 8mm Erythromycin (E 15) = R Penicillin (Pen.) = R

Nursyazmin Binti khairuddin (12273208031) Pathogenic Microbiology (HLB 22303)

**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 methicillin-resistant 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: As a conclusion, from this experiment the t he 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.