Diagnostic Techniques: Urine Culture

Transcription

1 Diagnostic Techniques: Urine Culture Melissa Schreiber, Presenter: Valencia College, Orlando, FL Objectives: After completing this project the students should be able to: 1. perform a routine urine culture using a simulated urine specimen inoculated with known bacteria; 2. isolate bacteria from these samples using differential and selective media; 3. identify the bacterial isolate using a battery of biochemical and screening tests used in previous exercises and in other parts of this exercise; 4. perform CFU counts on isolate and correlate clinical significance of this count as it pertains to the diagnosis of urinary tract infections (UTI); 5. recognize the relevance of routine urine culture in terms of the prevalence of UTI in the community as well as in hospital settings (nosocomial). Materials: Media: Mannitol Salt Agar (MSA); MacConkey Agar (MAC); Urea Broth (UB); Sulfide Indole Motility Medium (SIM) Staining Reagents and Glass Slides Volumetric Loop (1 L); Inoculating Loop; Inoculating Needle Cultures: Four unknowns labeled Unknown D, Unknown E, Unknown F, and Unknown G Techniques Needed: Aseptic Technique Dip and Swirl Technique for Urea Broth Straight Stabbing Technique for SIM Semi-Quantitative Method Bacterial Smear Preparation and Staining Microscopy Procedure: Day 1: Stain and Inoculation of MAC and MSA Plates 1. Based on the stain result, each student will label either a MAC or a MSA plate with Unknown D, E, F, or G and label the bottom according to standard laboratory practices. 2. Inoculate the MAC and MSA plate with the method that is demonstrated in the *Photographic Atlas on page 222 (Figure 7-11: Semi-Quantitative Method). 3. Incubate the MAC and MSA plates for 24 hours at 37 C. 4. Perform the Kirby Bauer method. See the following section. Day 2: Evaluation of MAC and MSA Plates and Inoculation of SIM and UB Tubes 1. Examine your MAC plates and note any characteristics in your lab manual (See Atlas page 13 and Observations and Results Table). Is there any growth on the plates and if so what color are the colonies? Refer to the Table of Culture Media in the Appendix. 2. Examine your MSA plates and note any characteristics in your lab manual (See Atlas page 14 and Observations and Results Table 6.6). Has the phenol red in the medium surrounding any of the colonies turned yellow? Refer to the Table of Culture Media found in the Appendix. 3. Count the number of colonies present on your MSA and MAC plate. Record your original cell density (OCD) (also known as bacterial concentration or density) for each plate and report your results in colony forming units abbreviated CFU per milliliter (CFU/mL). OCD = CFU / loop volume OCD = CFU / ml = CFU/10-3 Is your unknown sample s OCD higher than 100,000 CFU/mL? If your OCD is higher than 100,000 CFU/mL would this be a sufficient criterion for a UTI?

2 Inoculation of UB and SIM: 4. Each group will label 4 tubes of UB and SIM according to standard laboratory practices (class code; group number; name of medium; abbreviation of unknown D, E, F, G; student initials and date). 5. Select colonies from your MAC and MSA plates and inoculate 4 tubes of UB with each of the unknowns using the dip and swirl method. 6. Select colonies from your MAC and MSA plates and inoculate 4 tubes of SIM Agar deep with each of your unknowns using the straight stab method with your inoculating needle. 7. Incubate the UB and SIM tubes at 37 C for 24 hours. Day 3: SIM and UB Evaluations and Identification 1. Examine your UB tube for color changes and record your results in the Observations and Results section Table. Do you notice a color change in any tube(s)? Which one(s)? Refer to Atlas pages and Table of Culture Media found in the Appendix. 2. For your SIM tubes add 5 drops of Kovac s reagent. 3. Examine the tubes for H 2 S and indole production and observe for evidence of motility. Refer to Atlas pages for the H 2 S test; Atlas pages for the indole test; Atlas pages for the motility test. Also refer to the Table of Culture Media found in the Appendix. 4. Record your results in the Observations and Results Section Table. 5. Examine your results from the Stain, MAC and MSA plates, and UB and SIM tubes. Identify your organisms from the 4 unknowns by using the Appendix and record your interpretations in the Observations and Results Section Table. Observations and Results: Final Determinations of Unknown (Simulated) Urine Samples Specimen Stain Lactose (+) or (-) Mannitol (+) or (-) Urease (+) or (-) SIM H 2 S, Indole, Motility Identification Unknown D Unknown E Unknown F Unknown G Questions: 1. Describe a "clean catch" when obtaining a urine specimen. Why is a clean catch important? 2. If the numbers of organisms from a urine culture was 5.0 X 10 4 CFU per milliliter, is this a significant number of organisms for the individual to have an UTI? 3. What role does urease play in an UTI? What alkalinophilic bacteria are usually associated with this condition? 4. Name at least two bacterial species that are frequently implicated in urinary tract infections. 5. Describe personal hygiene practices that can lower your risk of an UTI. *Leboffe, Michael J., and Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratory. 4th ed. Englewood, CO: Morton Pub. Co., 2011.

3 Diagnostic Techniques: Kirby-Bauer Method Objectives: After completing this project the students should be able to: 1. perform antimicrobial susceptibility test by the disk diffusion technique otherwise known as the Kirby- Bauer Method; 2. understand the principle involved in this test and determine the factors that may affect the outcome of this test; 3. determine the suitability and efficacy of antimicrobials to positive and negative bacteria or both; 4. recognize the utility and relevance of these tests in the treatment and management of infectious diseases. Materials: Kirby-Bauer Method TSB culture of Unknown Trypticase Soy Agar (TSA) plates Antimicrobial Disks (Ampicillin, Ciprofloxacin, Gentamicin, and Sulfonamides) Sterile cotton swab Millimeter ruler Techniques Needed: Aseptic Technique Bacterial Lawn Procedure: 1. Each student will obtain 1 TSA plate with which to make a bacterial lawn from your unknown organism D, E, F, or G. 2. Soak a sterile swab into a well-mixed culture (gently tap the bottom of the tube) of your unknown for at least 5 seconds. 3. Squeeze out excess of the bacterial inoculum by pressing the swab around the mouth of the TSB tube. 4. Inoculate the TSA plate by swabbing the entire agar surface using close parallel lines from one edge of the plate to the other making sure not to leave any gaps. 5. Using the same swab (Do not dip it again in the culture!) swab the agar surface as above but in a direction perpendicular to the first lines of inoculation. 6. Repeat step 5, but this time swab the agar surface at a 45 angle (diagonally) to the first. 7. Finally, swab around the edge of the agar surface. 8. Dip a pair of forceps briefly in alcohol and flame it under the Bunsen burner. 9. Place the 4 different antimicrobial disks on each of the lawned TSA plates using the sterilized forceps. 10. Secure the disks on the agar surface by pressing them lightly using the sterilized forceps. 11. Incubate the plates in an inverted position at 37 C for 24 to 48 hours. Observations and Results: 1. After incubation, examine your TSA plates for zones of inhibition around each of the antimicrobial disks 2. Measure the diameter of the zones of inhibition using a millimeter ruler and record results. 3. Determine whether the test organisms are susceptible (S), intermediate (I) or resistant (R) to the antimicrobials on the TSA plate by consulting the Appendix. 4. Enter your results in the Observation and Results section Table.

4 Zone Diameters of Antimicrobials Tested Against the Unknowns and the Interpretations Organism Ampicillin (Zone=S, I, or R) Ciprofloxacin (Zone=S, I, or R) Gentamicin (Zone=S, I, or R) Sulfonamides (Zone=S, I, or R) Questions: 1. List each of the organisms used in the Kirby-Bauer Method and the antimicrobial that was most effective against each organism using this method. 2. If there are two antimicrobials that show an S result, which one will be your best choice and why? 3. What would the medical implication be if a Staphylococcus aureus strain was found to be resistant to ampicillin? 4. Did you note any colonies growing in the zone of inhibition? Why is this significant? 5. What test is done in the clinical setting that is similar to the Kirby-Bauer Method? 6. Refer to an outside source, and list the mode of action for each of the four antimicrobial drugs used in this test. 7. What are the side effects of each of the antimicrobials used in this test?

5 Diagnostic Techniques: Fecal Culture Objectives: After completing this project the students should be able to: 1. recognize members of the Enterobacteriaceae Group, with special emphasis on enteric pathogens such as Salmonella and Shigella; 2. perform routine stool culture and identify potential pathogens recovered from fecal samples; 3. utilize differential and selective media for the purpose of recovering these pathogenic bacteria; 4. understand the principles involved in each of the biochemical tests and interpret reactions exhibited in the test media used; 5. understand the applications of these biochemical and screening tests in terms of differentiating and identifying bacterial isolates in fecal samples. 6. understand the relevance of using stool culture as a diagnostic tool for gastroenteritis and food poisoning. Materials: Media: MacConkey Agar (MAC) Triple Sugar Iron Agar (TSI) Urea Broth (UB) Methyl Red and Voges-Proskauer Broth (MR-VP) Sulfide Indole Motility Media (SIM) Simmon s Citrate Agar (CIT) Inoculating Loop and Needle Four Cultures Labeled Unknown H, Unknown I, Unknown J, Unknown K Techniques Needed: Aseptic Technique Procedure: Day 1: Inoculation of MAC Plate 1. Each group will inoculate a MAC plate with Unknowns H, I, J, K. 2. For each group, divide your MAC plate in half and label with Unknown H, I, J, or K. Label the bottom according to standard laboratory practices (class code; group number; name of medium; student initials and date). 3. Inoculate the MAC plate by using a streaking technique demonstrated by the instructor. 4. Incubate the MAC plates for 24 hours at 37 C. Day 2: Evaluation of MAC Plates and Inoculation of TSI, UB, MR-VP, SIM, CIT Tubes 1. Examine your MAC plates and note any characteristics in your lab manual (See Atlas page 13 and Observations and Results Table). Is there any growth on the plates and if so what color are the colonies? Refer to the Table of Culture Media found in the Appendix. 2. Each group will label 4 tubes of TSI, UB, MR-VP, SIM, and CIT according to standard laboratory practices (class code; group number; name of medium; abbreviation of unknown H, I, J, or K; student initials and date). 3. Select colonies from your MAC plates and inoculate 4 tubes of UB and MR-VP with each of the unknowns using the dip and swirl method. 4. Select colonies from your MAC plates and inoculate 4 tubes of SIM Agar deep with each of your unknowns using the straight stab method with your inoculating needle. 5. Select colonies from your MAC plates and inoculate 4 tubes of CIT with each of the unknowns by using the fish tail method. 6. Select colonies from your MAC plate and inoculate 4 tubes of TSI Agar by stabbing the butt and streaking the slant using a heavy fish tail. Your instructor will demonstrate this inoculation technique. 7. Incubate the UB, SIM, CIT, and TSI tubes at 37 C for 24 hours. 8. Incubate the MR-VP tubes at 37 C for 48 hours.

6 Day 3: TSI, UB, MR-VP, SIM and CIT Evaluations and Identification TSI 1. Examine the TSI tubes for characteristic color changes and gas production. Be sure to include results for the slant and butt and indicate any gas or H 2 S production. Refer to the Appendix and pages of the Photographic Atlas. 2. Record your results using recommended symbols and abbreviations in the data tabulation portion of your laboratory report (Table). UB 1. Examine your UB tubes for color changes and record your results in the Observations and Results section Table. Indicate the tube(s) in which there is a color change. 2. See Atlas pages and refer to the Table of Culture Media found in the Appendix. MR-VP 1. For the MR-VP test, label a clean screw cap test tube with VP for the Vogues Proskauer test. Aseptically pipette 2.5 ml of MR-VP broth to the VP screw cap test tube. Broth should be turbid due to bacterial growth. 2. Label your original tube MR and use this tube for the Methyl Red test. 3. Methyl Red test: a. Add 5 drops of Methyl Red reagent to the tube labeled MR. b. Observe for red color change immediately. If the color does not change immediately or is a shade of yellow or orange, consider the result negative. See Atlas page 82. c. Record your results in the Observations and Results section Table. 4. Voges-Proskauer test: Use the screw cap tube for this test. a. Add 12 drops of VP Reagent A (α-naphthol) to the tube labeled VP. b. Add 4 drops of VP Reagent B (KOH) to the tube labeled VP. c. Shake the tube vigorously to oxygenate the medium. d. Allow the tube to stand for 10 minutes maximum for color development. e. If a rusty red color appears, the test is positive. If a copper green color appears the test is negative. Watch out for false positives that are colored copper brown. True VP positives are Bordeaux wine red in color. See Atlas page 98. f. Record your results in the Observations and Results section Table. SIM 1. For your SIM tubes add 5 drops or so of Kovac s reagent. 2. Examine the tubes for H 2 S and indole production and observe for evidence of motility. Refer to Atlas pages for the H 2 S test; Atlas pages for the indole test; Atlas pages for the motility test. Also refer to the Table of Culture Media found in the Appendix. 3. Record your results in the Observations and Results Section Table. CIT Examine your CIT tubes for color changes, see Atlas pages 64-65, and record your results in the Observations and Results section Table. OBSERVATIONS AND RESULTS: Identification of Unknown Fecal (Simulated) Samples Examine your results from the MAC plate; TSI, UB, MR-VP, SIM and CIT tubes. Identify your organisms from the 4 unknowns by using the Appendix and record your interpretations in the Observations and Results Section Table.

7 Table: Final Determinations of Unknown (Simulated) Fecal Samples Specimen MAC Plates TSI Tubes UB Tubes MR-VP Tubes SIM Tubes CIT Tubes Identification Unknown H Unknown I Unknown J Unknown K Questions: 1. IMViC a. What does the term IMViC mean? b. Why is the IMViC useful in identifying Enterobacteriaceae? c. Are further biochemical tests needed for complete identification? 2. In the MR-VP test, what end product(s) are detected in the following? a. Mixed acid fermentation b. Neutral fermentation 3. What is meant by the term enteric pathogen? 4. What is the value of serological identification of a microorganism as compared with culture identification? 5. Why is it not necessary to collect a stool for culture in a sterile container? 6. What diseases are caused by Salmonella, Shigella, and Escherichia coli 0157:H7? 7. How does intestinal flora gain entry to the body?

8 Appendix Table of Culture Media Abbreviation Purpose C Special Ingredients Preparation Inoculation Reading Criteria MacConkey (MAC) Agar Mannitol Salt Agar (MSA) Methyl Red Voges-Proskauer Broth(MR-VP) Simmon s Citrate Agar (CIT) Sulfide Indole Motility Medium (SIM) Isolation of -negative Enterics Isolates and differentiates Staph species Two separate tests to determine what end products result when glucose is metabolized Detection of citrate utilizers Screening for H 2 S, Indole production, and motility S & D S & D B SA = Bile Salts & Crystal Violet; DA =Lactose; ph Indicator = Neutral Red SA = 7.5% NaCl; DA = Mannitol; ph Indicator = Phenol Red MR Test: Reagent = Methyl Red; VP Test: Reagents = alphanaphthol and KOH B Citrate Carbon; Ammonium Phosphate - Nitrogen; ph Indicator = Bromthymol Blue B Cysteine = H 2 S production Reagent = Kovac s Reagent Typical Typical After incubation MR-VP broth is split into two tubes - MR test; VP test Quadrant Streak Quadrant Streak Dip & Swirl 1. Purple growth= lactose fermentation + 2. Colorless growth = lactose fermentation 1.Growth, medium is lemon-yellow = mannitol fermentation + 2. Growth, medium is pink = mannitol fermentation- 3. No growth = Staphylococcus 1. MR Test cherry red = MR + = mixed acid fermentation +; Not red = MR - = mixed acid fermentation - 2. VP Test rusty red = VP+ = neutral fermentation +; Not red = VP- = neutral fermentation Agar Slant Fish Tail 1. Blue = citrate + 2. Green = citrate Semi-solid agar Stab 1. Black ppt = H 2 S+ 2. Add Kovac s Reagent, if Red = Indole + 3. Cloudiness throughout the test tube = Motility + Triple Sugar Iron (TSI) Screening for fermentative ability of glucose and lactose &/or sucrose B ES = Glucose, Lactose, Sucrose ph Indicator = Phenol Red Cysteine = H 2 S production Short slant, large butt Critical: Stab butt; heavy fish tail on slant 1. Slant: red = alkaline = K, yellow = acid = A 2. Butt: red = K, yellow or black = A 3.Butt gas: (+) 4. Butt black: H 2 S (+)

9 Abbreviation Purpose C Special Ingredients Preparation Inoculation Reading Criteria Trypticase Soy Agar (TSA) Trypticase Soy Broth (TSB) Urea Broth (UB) Growth of wide range of bacteria Growth of wide range of bacteria Production of the exoenzyme urease G None Typical Varies Growth of wide range of bacteria; making smears & lawns G None Typical Dip & Swirl Growth of wide range of bacteria; making B Urea as substrate ph Indicator = Phenol Red Filter sterilized Only Dip & Swirl smears & lawns 1. Orange = urease (-) 2. Fuchsia = urease (+) Legend: Category (C): B = Biochemical D = Differential S = Selective G = General Special Ingredients: ES = Energy Source; DA = Differential Agent ; SA = Selective Agent; SP = Selective Property Appendix Isolation and Identification of Urine Pathogens Bacterial species Stain Shape UB (Urease) MSA Mannitol Fermentation MAC Lactose Fermentation SIM Staphylococcus epidermidis (+) Coccus Urease (+) (-) No Growth -,-,- Staphylococcus saprophyticus (+) Coccus Urease (+) (+) No Growth -, -, - Escherichia coli Proteus vulgaris (-) (-) Rod Urease (-) No Growth (+) -,+,+ Rod Urease (+) No Growth (-) +,+,+

10 Appendix Isolation and Identification of Fecal Pathogens Bacterial species MAC Lactose Fermentation TSI UB (Urease) MR-VP SIM CIT Salmonella enteritidis (-) K/A + + Urease (-) +/- +,-,+ + Shigella sonnei (-) K/A - - Urease (-) -/+ -,-,- - Escherichia coli (+) A/A + - Urease (-) +/- -,+,+ - Proteus vulgaris (-) A/A - + Urease (+) +/- +,+,+ - Appendix Zone Diameter Interpretive Standards Antimicrobial Agent Class of Antimicrobial Disk Content Resistant ( R ) Intermediate ( I ) Susceptible ( S ) Ampicillin Beta-lactam 10 ug Ciprofloxacin Fluoroquinolone 5 ug Gentamicin Aminoglycoside 10 ug Sulfadiazine Sulfonamides ug MIC values recommended by the Clinical and Laboratory Standards Institute (CLSI)