Diagnostic Techniques: Urine Culture Melissa Schreiber, Presenter: Valencia College, Orlando, FL
[email protected] 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) Gram Staining Reagents and Glass Slides Volumetric Loop (1L); 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 Gram Staining Microscopy Procedure: Day 1: Gram Stain and Inoculation of MAC and MSA Plates 1. Based on the Gram 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 37C. 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 -3 OCD = CFU / 0.001 mL = CFU/10 _____________________________________________________ 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?
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 96-97 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 H2S and indole production and observe for evidence of motility. Refer to Atlas pages 93-94 for the H2S test; Atlas pages 74-75 for the indole test; Atlas pages 82-83 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 Gram 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
Gram Stain
Lactose (+) or (-)
Mannitol (+) or (-)
Urease (+) or (-)
SIM H2S, 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? 4
2. If the numbers of organisms from a urine culture was 5.0 X 10 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.
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 KirbyBauer 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 Gram positive and Gram 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 37C 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.
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?
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 37C. 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.
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 2S production. Refer to the Appendix and pages 95-96 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 96-97 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 H2S and indole production and observe for evidence of motility. Refer to Atlas pages 93-94 for the H2S test; Atlas pages 74-75 for the indole test; Atlas pages 82-83 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.
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?
Appendix – Table of Culture Media
Abbreviation
Purpose
C
MacConkey (MAC) Agar
Isolation of Gram-negative Enterics
S & D
Mannitol Salt Agar (MSA)
Isolates and differentiates Staph species
Methyl Red Voges-Proskauer Broth(MR-VP)
Special Ingredients
Preparation
Inoculation
Reading Criteria
SA = Bile Salts & Crystal Violet; DA =Lactose; pH Indicator = Neutral Red
Typical
Quadrant Streak
S & D
SA = 7.5% NaCl; DA = Mannitol; pH Indicator = Phenol Red
Typical
Quadrant Streak
Two separate tests to determine what end products result when glucose is metabolized
B
MR Test: Reagent = Methyl Red; VP Test: Reagents = alphanaphthol and KOH
After incubation MR-VP broth is split into two tubes - MR test; VP test
Dip & Swirl
Simmon’s Citrate Agar (CIT)
Detection of citrate utilizers
B
Agar Slant
Fish Tail
Sulfide Indole Motility Medium (SIM)
Screening for H2S, Indole production, and motility
B
Citrate – Carbon; Ammonium Phosphate Nitrogen; pH Indicator = Bromthymol Blue Cysteine = H2S production Reagent = Kovac’s Reagent
1. Purple growth= lactose fermentation + 2. Colorless growth = lactose fermentation – 1.Growth, medium is lemon-yellow = mannitol fermentation + 2. Growth, medium is pink = mannitol fermentation3. 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 – 1. Blue = citrate + 2. Green = citrate –
Semi-solid agar
Stab
1. Black ppt = H2S+ 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 = H2S 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: H2S (+)
Abbreviation
Purpose
C
Special Ingredients
Preparation
Inoculation
Reading Criteria
Trypticase Soy Agar (TSA)
Growth of wide range of bacteria
G
None
Typical
Varies
Trypticase Soy Broth (TSB)
Growth of wide range of bacteria
G
None
Typical
Dip & Swirl
Urea Broth (UB)
Production of the exoenzyme urease
B
Urea as substrate pH Indicator = Phenol Red
Filter sterilized Only
Dip & Swirl
Growth of wide range of bacteria; making smears & lawns Growth of wide range of bacteria; making 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
Gram Stain
Shape
UB (Urease)
MSA Mannitol Fermentation
MAC Lactose Fermentation
SIM
Staphylococcus epidermidis
Gram (+)
Coccus
Urease (+)
(-)
No Growth
-,-,-
Staphylococcus saprophyticus
Gram (+)
Coccus
Urease (+)
(+)
No Growth
-, -, -
Escherichia coli
Gram (-)
Rod
Urease (-)
No Growth
(+)
-,+,+
Proteus vulgaris
Gram (-)
Rod
Urease (+)
No Growth
(-)
+,+,+
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
≤ 13
14-16
≥ 17
Ciprofloxacin
Fluoroquinolone
5 ug
≤ 15
16-20
≥ 21
Gentamicin
Aminoglycoside
10 ug
≤ 12
13-14
≥ 15
Sulfadiazine
Sulfonamides
250-300 ug
≤ 12
13-16
≥ 17
MIC values recommended by the Clinical and Laboratory Standards Institute (CLSI)
