Diagnostic Techniques: Urine Culture

Melissa Schreiber, Presenter: Valencia College, Orlando, FL

mschreiber@valenciacollege.edu
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 samples 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 Kovacs 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)
Simmons 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 (-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 Kovacs 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

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

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

Simmons Citrate
Agar (CIT)

Detection of
citrate utilizers

Agar Slant

Fish Tail

Sulfide Indole
Motility Medium
(SIM)

Screening for
H2S, Indole
production, and
motility

Citrate
Carbon;
Ammonium
Phosphate Nitrogen;
pH Indicator =
Bromthymol
Blue
Cysteine = H2S
production
Reagent =
Kovacs
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 Kovacs
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

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

Special
Ingredients

Preparation

Inoculation

Reading Criteria

Trypticase
Soy Agar (TSA)

Growth of wide
range of bacteria

None

Typical

Varies

Trypticase
Soy Broth (TSB)

Growth of wide
range of bacteria

None

Typical

Dip & Swirl

Urea Broth (UB)

Production of the
exoenzyme
urease

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)