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

LAB REPORT 8: FECAL SAMPLE Objectives: 1. To observe the presence of vibrio spp. in the stool sample. 2. To identify the microorganisms that causing the infection to stool sample. 3. To determine the microorganisms are sensitive to which antibiotics.

Introduction: The bacteria in the stool are representative of the bacteria that are present in the gastrointestinal tract. The human GIT contain normal flora which consist of bacteria and fungi. The balance of normal flora maybe interrupted through consumption of, for example broad-spectrum of antibiotics which will inhibit the growth of normal flora and allow bacteria resistant to the antibiotic to survive. Pathogenic bacteria enter human body when someone eats food or drinks water that has been contaminated. This may include raw or undercooked eggs, poultry or beef, unpasteurized milk, and contaminated water from lakes. The most common symptoms of a pathogenic bacterial infection are prolonged diarrhea, bloody diarrhea, mucus in the stool, abdominal pain, and nausea. If diarrhea lasts more than a few days, it may lead to dehydration and electrolyte imbalance. A stool test will be done to detect and identify the pathogenic bacteria in the stool. In the laboratory, the test is initiated by applying a small amount of a fresh faecal sample to a variety of nutrient media, such as TCBS agar and McConkey agar. Thiosulfate Citrate Bile Salts Socrose (TCBS) agar is used for the selective isolation of Vibrio cholera, Vibrio parahaemolyticus and other types of vibrio from a variety of clinical and nonclinical specimens. Inhibition growth of gram-positive bacteria in this agar is achieved by the incorporation of oxgall, which is a naturally occurring substance containing a mixture of bile salts, and sodium cholate, a pure bile salt. Sodium thiosulfate serves as a sulfur source and, in combination with ferric citrate, detects hydrogen sulfide production. Sucrose is included as a fermentable carbohydrate for the metabolism of vibrios. The alkaline pH of the medium enhances the recovery of Vibrio cholerae. Thymol blue and bromthymol blue are included as indicators of pH changes. TCBS is the primary plating medium universally used for the selective isolation of vibrios that cause cholera, diarrhea and food poisoning. It was developed by Kobayashi et al. who modified the selective medium of Nakanishi. The combination of alkaline peptone water and TCBS Agar is used in many procedures for the

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

isolation of V. cholerae and other Vibrio species from faeces. Vibrio choleraeis a gram negative comma shaped bacterium with a polar flagellum that causes cholera in humans while Vibrio parahaemolyticus is a curved, rod-shaped, Gram-negative bacterium found in brackish saltwater, which, when ingested, also causes gastrointestinal illness in humans. V. parahaemolyticus is oxidase positive, facultatively aerobic, and does not form spores. Like other members of the genus Vibrio, this species is motile, with a single, polar flagellum. McConkey agar is a culture medium designed to grow Gram-negative bacteria and stain them for lactose fermentation. It contains bile salts, crystal violet dye, neutral red dye, lactose and peptone. In this experiment, the stool sample was cultured on TCBS and McConkey agar. Then it was observed under microscope for detection present of any vibrio sp.. Then a few biochemical tests were run to observed its reaction. Lastly, sensitivity test was run to measured the sensitivity of the growth bacteria towards which types of antibiotics. Materials: 1. Fecal sample 2. McConkey agar 3. TCBS agar 4. Mueller Hinton agar 5. Microscope 6. Slides 7. Wire loop 8. Bunsen burner 9. Grams stain 10. Microscope 11. Toothpick 12. Oxidase reagent 13. Sterile swab 14. Peptone water 15. Broth for MR/VP 16. TSI agar 17. Motility test medium 18. Citrate medium 19. Methyl red reagent 20. Voges-proskauer reagent 21. Kovacs reagent (Indole) 22. Dropper 23. Antibiotics discs: a) Cerfuroxime (CXM 30) b) Gentamicin (CN 10) c) Ampicillin (AMP 10) d) Augmentin (AMC)

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

Method: Day 1; 1. The stool sample from the container was observed macroscopically whether it solid, semi solid or watery stool and also the color of the stool. 2. Then, the microbes were cultured on the TCBS agar and McConkey agar. Then it was incubate for 24 to 48 hours in the incubator at temperature of 37C.

Day 2; 1. After 24 to 48 hours incubation, the cultured microbes on the TCBS and McConkey agar were observed it culture characteristics and whether it ferments lactose or not. The numbers of colony in the cultured were calculated. 2. Gram stained was done and was seen under microscope to see its shape and arrangements. 3. Then, a few biochemical tests were run to observed it reactions based on the observation whether it is Gram negative or Gram positive. 4. The biochemical test that was run for this experiment were TSI test, motility test, indole test, MR test, VP test and citrate test. The entire tests were run for every colony that was found. 5. Oxidase test was also run since there are growths of non-ferment lactose in the McConkey agar. 6. For oxidase test, the cultured microbes were put on a litmus paper that contains a few drop of oxidase reagent and the result of color changes on litmus paper was observed and recorded. 7. For catalase test, the cultured microbes were mixed together with the catalase reagent and the result of production of bubbles was observed and recorded. 8. For TSI test, the cultured microbes were stab using straight wire loop until the bottom of the butt of TSI agar and striking at the surface of the slant. Then it was incubated for 24 hours at 37C.

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

9. For motility test, the cultured microbes were stab about three quarter of the tube. Then it was incubated for 24 hours at 37C. 10. For indole test, the cultured microbes were mixed into the peptone water tube. Then it was incubated for 24 hours at 37C. 11. For MR and VP test, the cultured microbes were mixed with the MR-VP media. Then it was incubated for 24 hours at 37C. 12. For citrate test, the cultured microbes were striking on the surface of citrate agar. Then it was incubated for 24 hours at 37C. Day 3; 1. After 24 hours, the results of TSI test, motility test and citrate test were observed and recorded. 2. For the indole test, MR test and VP test, a few procedures were done to obtain results. 3. For indole test, the cultured media were added with 5 drops of Kovacs reagent. The changes in the tube were observed and recorded. 4. For MR test, the cultured media were added with 5 drops of MR (methyl red) reagent. The changes were observed and results were recorded. 5. For VP test, the cultured media were added with 6 drops of VP 1 and 2 drops of VP 2. It was let mixed for 2 minutes. Then the changes were observed and results were recorded. 6. After all the data were obtained, the results were compared with the biochemical reactions table that was given earlier and the type of microorganisms was identified. Result: Microscopic view:

Types = Gram negative bacilli (McConkeys agar)

Biochemical test:-

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

Oxidase = Negative Indole = Negative MR = Positive VP = Negative Citrate = Negative Motility = Motile TSI = Slant / Butt / Gas Alkaline (red) / Acid (yellow) / No gas (black precipitate form)

Sensitivity test: Discussion: From this experiment, the observed colony culture from TCBS agar plate, there is only a little growth of bacteria which showed no significant growth. After stain with Grams stain, the observation showed that the bacteria is Gram positive which indicated that this is not the bacteria that been looking for. TCBS agar only used to growth Gram negative Vibrio sp.. So this proved that this stool sample does not have any Vibro sp. in it and this patient have infection from other types of bacteria. Sensitivity test were not run for this bacteria because its not the cause of infection. Meanwhile in McConkey agar plate, the colonies isolated from this agar showed non-lactose ferment bacteria. After stained with Grams stain, the observation under microscope showed Gram negative bacili. From this observation, biochemical test was run to determine which species the bacteria are. Since isolated colonies on the McConkey agar are non-lactose ferment, oxidase test were run and it showed negative results. IMViC test were also run, the result showed negative for indole, citrate and VP test and positive for MR test. The bacteria are motile in the motility test. In the TSI test, the slant of the TSI agar is alkaline, the butt is Cerfuroxime (CXM 30) = 18mm Gentamicin (CN 10) = 25mm Ampicillin (AMP 10) = 20mm Augmentin (AMC) = 23mm

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

acidic and it does not produce any hydrogen gas however it does form black precipitate of H2S. Since this bacteria is Gram negative on McConkey agar, sensitivity test were run using four different types of antibiotics. There are Cerfuroxime (CXM 30) , Gentamicin (CN 10), Ampicillin (AMP 10) and Augmentin (AMC). The results of the sensitivity test are Cerfuroxime (18mm), Gentamicin (25mm), Ampicillin (20mm) and Amoxicillin (23mm). From these results, it showed that these bacteria are very sensitive towards Gentamicin, moderately sensitive towards Augmentin and only a bit sensitive towards Cerfuroxime antibiotics. So this patient should be prescribed with Augmentin for seven days to kill the bacteria because its moderate sensitivity towards the bacteria will not be harmful to human. Conclusion: As a conclusion, from this experiment the bacterias colony that was isolated from McConkey agar was identified as Salmonella sp. Further test need to be done to identify the specific types of the bacteria.