Lab 7: Approach to Identification – Bacteriology of the Gastrointestinal Tract

1. Lab 7: Approach to Identification – Bacteriology of the Gastrointestinal Tract

2. Overview: The Gastrointestinal Tract For more information on the Gastrointestinal Tract see your handout.

3. The Gastrointestinal Tract: Host Defense Mechanisms Anatomical characteristics/mechanisms to prevent/fight infection: • High acidity of stomach prevents most microbes from surviving, making it almost sterile. • Normal microbiota – a variety of microorganisms that compete with each other for space and nutrients, aid in digestion, immunity and preventing infection from pathogens. • Normal flora - includes Enterobacteriaceae, Staphylococcus, Streptococcus, Enterococcus and various anaerobic bacteria. See handout for more details.

4. Gram-negative Enterobacteriaceae • Enterobacteriaceae are a family of gram-negative bacteria that include both harmless and pathogenic bacteria: • Pathogenic Enterobacteriaceae include Salmonella, Shigella, Enterobacter, E. Coli, Klebsiella, Proteus vulgaris… • Enterobacteriaceae are commonly found as normal flora of the gastrointestinal tract (termed “enterics”) but are also common causes of urogenital infections (E. coli, Proteus vulgaris, Enterobacter…) E. coli

5. The Gastrointestinal Tract:When things go wrong… Colitis Gastroenteritis Cholera See handout for more details.

6. The Gastrointestinal Tract: Specimens & Diagnosis • Stool is added to a container with liquid Cairy-Blair transport medium. • Upon receipt the laboratory will perform special rapid antigen tests to detect pathogens or toxins. • A stool culture may be performed as well; results can take 3-4 days. • Gram stain for white blood cells and predominance of thin GNRs that are curved - this indicates Campylobacter infection. • Ova and Parasites- Special transport and preservation media is required. See handout for more details.

7. How do we identify the microorganism causing the infection? Oxidase test Gram stain MacConkey Agar LABORATORY TESTING!

8. Identification Flowchart

10. Many different biochemical tests to be reviewed/performed today: • REVIEW - Oxidation-Fermentation Glucose Agar • REVIEW – Oxidase • REVIEW – Indole • Phenol Red Broth (Carbohydrate fermentation) • Triple Sugar Iron Agar • Urine Culture Take your time, stay organized, and be sure to label correctly.

11. REVIEW:Oxidative-Fermentative Media • O-F media contains a single carbohydrate (glucose) and a pH indicator (bromthymol blue) • The color of the media changes such that it turns yellow below pH 6, blue above pH 7.6, and green from pH 6-7.6 • A layer of oil is added to exclude air (distinguishes between the two metabolic pathways).

12. REVIEW: Oxidase Test • Oxidase is a specific enzyme used in by certain bacteria in cellular respiration (metabolism). • This test, like the catalase test, is used to determine if the bacteria has a specific enzyme, oxidase. • An oxidase reagent is added to the sample of bacteria and if oxidase is present, there will be a color change (purple = a positive result).

13. REVIEW: SIM/Indole Test • The indole test is used to determine if the bacteria produce an enzyme called tryptophanase, which responsible for producing the end product indole. • It is also used to determine if sulfide is being produced (another identifying trait of certain bacteria) and if the bacterium is motile. • SIM (sulfide, indole, motility) medium and a special reagent called Kovac’s reagent is used for this test. • This is a multipurpose medium that can evaluate 3 different physiological characteristics (sulfide production, indole production, and motility) in one test!

15. Phenol Red Broth/Carbohydrate Fermentation • Fermentation of carbohydrates by bacteria results in the production of an acid and, in some cases, a gas. • Bacteria differ in that they can only ferment certain types of carbohydrates. • Therefore, fermentation results can be used to identify bacteria.

16. Carbohydrate Fermentation Media (Phenol Red Broth) • Differential medium: uses metabolic differences to distinguish and identify bacteria e.g. carbohydrate fermentation tubes.

17. Carbohydrate Fermentation Media (Phenol Red Broth) Each carbohydrate fermentation tube contains the following: • Carbohydrate: Each broth contains a single fermentable carbohydrate (glucose, lactose, sucrose). • Peptone: protein derivatives (other food for bacteria) • Phenol-Red: This is a pH indicator that is RED at pH 7 or higher (alkaline) but turns YELLOW at low pH (acidic). The broth is initially pH neutral. If fermentation occurs, and acid by-products are formed, the solution will turn yellow. • Durham Tube: This is a small test tube that is inverted (upside-down) in the broth. If gases are produced, they will be trapped in the Durham Tube and a gas bubble will form.

18. Carbohydrate Fermentation Media (Phenol Red Broth) • Thus 2 things can be determined from carbohydrate fermentation tubes: 1. Can the species ferment the carbohydrate or not? 2. If the species can ferment the carbohydrate, is it producing gas?

19. Phenol Red Broth Lab ProcedurePages 94-95

20. Triple-Sugar Iron Agar • Triple-Sugar Iron agar is a differential medium used to identify gram-negative enteric bacteria based on their ability to ferment selected carbohydrates and produce hydrogen sulfide (a metabolic by-product). • Differential because it contains: • three sugars (glucose, sucrose, lactose) • a pH indicator (phenol red) • two sources of sulfur (cysteine and thiosulfate) • an iron salt (used to detect the production of sulfur) • peptone

21. Review: Phenol Red Broth • Phenol red broth was used previously to detect fermentation of a single carbohydrate. • Unlike PR broth, TSI agar contains three sugars: glucose, sucrose, and lactose. • Organisms usually utilize glucose first. Once it is exhausted bacteria will switch to one of the other sugars (if possible). Phenol red broth

22. TSI Agar – Deep Slant • TSI agar is dispensed into test tubes to produce a slant with a deep butt (bottom of the tube). • The medium is inoculated by stabbing into the butt with an inoculating needle to introduce the bacteria to deep within the butt (low oxygen).

23. Triple Sugar Iron Agar Lab Procedure: Part 1Pages 99-100

24. Urine Culture Lab: Part 1 Step 1 Step 2 Please see the handout for detailed procedure.

25. Assignments Lab Reports: • Phenol Red Broth – Review questions 1-3 • Triple Sugar Iron agar – Questions 1-3 • Lab Smart (reminder): • Identification of Unknown Bacteria • – Available Oct 21st, Due Nov 17th

26. Phenol Red Broth Lab Procedure: Part 2 • Examine tubes for color change and gas production and report your findings in your lab report (sketch your results).

27. Triple Sugar Iron Agar Lab Procedure: Part 2

28. Triple Sugar Iron Agar Lab Procedure: Part 2

29. Urine Culture Lab: Part 2 In part 2 of the lab we will determine the concentration of the organisms present in each urine specimen. Calculation 0.001ml = 1ul (which is 1/1000 of an ml) Each colony = 1 colony forming unit or CFU CFU/ml = 1000 x the number of colonies growing on the plate surface