CLINICAL BACTERIOLOGY 1

Infections of the Urinary Tract and Genital Tract Infections

LEC 17 BY PICHI ARCILLE ENARDECIDO MALINTAD
November 20, 2022

LESSON OUTLINE ○ All areas of the urinary tract above the urethra in a
healthy person are sterile.
○ Only the distal portion is colonized by a large
1. INFECTIONS OF THE URINARY TRACT AND GENITAL number of microflora
TRACT : URINARY TRACT INFECTIONS ● The presence of bacteria in the urine is called bacteriuria
1.1. Review of the Anatomy of the Urinary Tract System ○ This is not an indication of UTI unless the colony
1.2. Resident Microbiota of the Urethra count of the bacteria is significant
1.3. Epidemiology and Pathogenesis ○ That is why in the lab when we do urine culture, we
1.4. Factors that Favor UTI do a quantitative count to discriminate between
1.5. Pathophysiology of UTI contamination, colonization, and infection.
1.6. Etiologic Agents
1.7. Route of Infection
1.8. Types of UTI
1.9. Specimen Collection
1.9.1.1. Clean-Catch Midstream Urine
1.9.1.2. Straight Catheterized Urine
1.9.1.3. Suprapubic Bladder Aspiration
1.9.1.4. Indwelling Catheter
1.10. Specimen Transport
1.11. Microscopic Examination (Screening Test)
1.11.1.1. Gram Staining
1.12. Chemical Methods
1.12.1.1. Indirect Indices
1.12.1.2. Nitrate Reductase (Greiss Test) Figure 1. Human Urinary System
1.12.1.3. Leukocyte Esterase Test
1.12.1.4. Catalase : UPI Uriscreen
1.13. Automated and Semi Automated System RESIDENT MICROBIOTA OF THE URETHRA:
1.13.1.1. Yellow IRIS System The presence of these organisms is always an indication of
1.13.1.2. Vitek System contamination
1.13.1.3. BAC-T-Screen 2000 ● Coagulase-negative staphylococci
1.14. Drawbacks of Screening Procedures ○ excluding Staphylococcus saprophyticus
1.15. Methods of Culturing Urine and Interpretative Criteria ● Viridans and nonhemolytic streptococci
1.16. Inoculation and Incubation of Urine Cultures ● Lactobacilli (adult females)
1.17. Classical Method of Interpreting Urine Culture ● Diphtheroids (Corynebacterium spp.)
1.18. General Interpretative Guidelines for Urine Culture ● Nonpathogenic (saprobic) Neisseria spp. (adult women)
2. INFECTIONS OF THE URINARY TRACT AND GENITAL Anaerobic cocci
TRACT : GENITAL TRACT INFECTIONS ● Propionibacterium spp. (adult patients)
2.1. Bacterial Flora of Genital Tract ● Commensal Mycobacterium spp.
2.2. Pathogenic Organisms Recovered from the Genital ● Commensal Mycoplasma spp.
Tract ● Yeasts (pregnant, adult females)
2.3. GIT and Systemic Pathogens as STD Agents
2.4. Laboratory Diagnosis of GIT’s : Lower GIT
2.5. Specimen Collection EPIDEMIOLOGY & PATHOGENESIS
2.5.1.1. Urethral Discharge
2.5.1.2. Urogenital Swab In Females
2.5.1.3. Cervical/Vaginal ● Incidence of UTI is more common in females than in males
2.6. Swabs for Isolation of Gonococci ● Shorter urethra
2.7. Swabs for Isolation of Chlamydia/Mycoplasma ○ Bacteria can reach the bladder more easily
2.8. Direct Microscopic Examination ● Proximity of the urethra to the anus
2.9. Bacterial Vaginosis ○ Lies in close proximity to the warm moist perirectal
2.10. Processing Urethral Specimens Suspected to Contain region
Gonococci ● Sexual activity
2.11. Identifying Neisseria ● Increases during pregnancy
○ Incidence of bacteriuria increases as a result of
anatomic and hormonal changes that favor the UTI
INFECTIONS OF THE URINARY TRACT AND GENITAL TRACT development (can lead to serious infections in both
the mother and fetus)
REVIEW OF THE ANATOMY OF THE URINARY TRACT SYSTEM ● Changes in the GUT mucosa related to menopause
○ Estrogen deficiency in post-menopausal usually
Consists of the following: Kidney, Ureters, Bladder, and Urethra results in the decrease of a normal vaginal
microbiota (e.g. presence of Lactobacilli) which is
● The urine from the ureter and bladder is sterile under normal associated with a recurrent UTI in females
conditions. Once voided, urine passes over the superficial
urogenital membranes and becomes contaminated by the In Males
normal flora of these areas. ● During the first year of life, UTI occur in less than 2% in males
● It is the urethra that has a resident microflora that colonize and females
the epithelium in the distal portion. ● Incidence is extremely low after age 1 until age 60.
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● Enlargement of the prostate
❖ Interferes with the emptying of the bladder which
leads to bacterial prostatitis.
● Bacterial prostatitis
FACTORS THAT FAVOR UTI (Males and Females)
1. Any anatomic barrier to free the flow of urine thru the UT
● Example: presence of kidney stones
● It will prevent the flow of urine to the urinary tract
2. Previous infection with certain urea-splitting organisms
(Proteus) → associated with formation of urinary stones
which predisposes the patient to infection.
● Rationale: Proteus is able to hydrolyze urea via
urease production so hydrolysis of urea will result to
increase in pH
● Once you have an increase in pH, the urine would
be alkaline. This is toxic to the kidney cells and
also stimulates the formation of kidney stones.
3. Introduction of foreign body into the UT
● Especially the ones that remains in place for a long
time
● Example: use of a foley catheter; as many as 30%
of hospitalized patients who receive short term
catheterization develop UTI.
PATHOPHYSIOLOGY OF UTI
1. Uncomplicated UTI
● Bacterial or yeast infection in a structurally and
neurologically normal urinary tract
2. Complicated UTI
● Bacterial or yeast infection in a urinary tract with
functional or structural abnormalities.
ETIOLOGIC AGENTS
We call them uropathogens since they cause UTI. They can be
divided into three.
1. Uncomplicated Community acquired UTI
● Occur primarily in sexually active young women with
normal GU tracts and no prior instrumentation and
are usually caused by antibiotic-susceptible
bacteria.
a. E. coli (UPEC)-most frequent cause
● At a molecular level, E. coli is
designated as a uropathogenic
E, coli (UPEC) since it causes
UTI and it is different from other
types of E. coli like the E. coli
025H4 which has recently
emerged as a significant urinary
tract pathogen in community
acquired infections.
b. Klebsiella species
c. Other enterobacteriaceae
d. S. saprophyticus
2. Complicated UTI
● Occur in individuals who have one or more structural
or functional genitourinary (GU) abnormalities or
have indwelling catheters and whose conditions
cannot be controlled with therapy
● Very common in patients with recurrent infections
a. Proteus
b. Pseudomonas
c. Klebsiella spp.
d. Enterobacter spp.
3. Hospital-acquired UTI
● Hospitals or healthcare environments play an
important role in determining the organisms involved
in UTIs.
● Hospitalized patients are most affected by:
a. E. coli
b. Klebsiella spp.
c. Staphylococci
d. Other Enterobactericiae
e. P. aeruginosa
f. Enterococci
ROUTE OF INFECTION
Bacteria can invade and cause UTI via 3 major routes - ascending
route, hematogenous route, and lymphatic pathways.
1. Ascending route: most common route or common course of
infection in females
○ UTIs are more often in females because of their
short urethra and its proximity to the anus,
increasing bacterial contamination of the female
urethra.
○ For UTIs to occur in the ascending pathway, the
Enteric gram (-) bacteria and other microorganisms
that originate in the GIT must be able to colonize the
vaginal cavity or the periurethral area.
■ Once these organisms gain access to the
bladder, they may multiply and go up to the
ureters then to the kidneys.
2. Descending route: hematogenous route (blood-borne route)
○ Hematogenous spread usually occurs as a result of
bacteremia.
○ Any systemic infection can lead to shedding of the
kidney.
○ Certain organisms such as S. aureus and
Salmonella spp. are particularly invasive.
○ Most infections involving the kidneys are acquired
through ascending route, but there are some who
are not (mentioned in square bullets below).
○ Organisms which often indicated pyelonephritis via
hematogenous route
■ Yeasts usually C. albicans
■ M. tuberculosis
■ Salmonella sp.
■ Leptospira spp.
■ S. aureus
○ Since the presence of any of these uropathogens is
an indication of pyelonephritis, it should be reported
right away to the clinician.
TYPES OF UTIs:
1. Urethritis
● Infection of the urethra
● Symptom associated with urethritis is dysuria
(painful or difficult urination and frequency).
● Inflammation of the urethra, presenting as dysuria
and discharge are considered to be STDs.
● Other causes include trauma, allergic, or chemical.
● A common infection sometimes considered as a
sexually transmitted disease.
2. Cystitis
● Inflammation of the bladder, presenting dysuria,
urinary frequency and urgency – meaning, there is a
compelling need to urinate. It is often due to gram
(-) bacilli such as E. coli, Proteus, and Klebsiella. It
occurs more frequently in women than men.
● The symptoms are not only resulted from the
inflammation of the bladder but also due to the
multiplication of bacteria in the urine and urethra.
● Most often, there is tenderness and pain over the
area of the bladder.
● In some individuals, the urine is grossly bloody so
the patient may note urine cloudiness and a bad
odor.
● Since cystitis is a localized infection, fever and other
signs of systemic illness are usually not present.
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3. Acute urethral syndrome
● Patients with this syndrome are primarily young,
sexually active females, who experience dysuria,
frequency, and urgency.
● (can be diagnosed with the following:) Defined as
more than 8 leukocytes/cumm of uncentrifuged urine
or approximately 2-5 leukocytes/HPO in centrifuges
urine sediment but yield fewer organisms than 105
colony-forming units of bacteria per milliliter
(CFU/mL) urine on culture.
4. Pyelonephritis
● Infection in the kidney.
● This is due to infection in the lower tract ascending
to the kidney (specifically on the kidney
parenchyma, the calyces – which is the cup-shaped
division of the renal pelvis, and the pelvis – which is
located at the upper end of the ureter that is located
inside the kidney and is usually caused by bacterial
infection).
● Symptoms include fever, chills, nausea, vomiting,
and lower back tenderness, as well as dysuria.
○ Verbatim in the lec includes: fever and
flank (lower back pain), frequently lower
tract symptoms. There is also frequency,
urgency, and dysuria.
○ Patients will also exhibit systemic signs of
infection such as vomiting, diarrhea, chills,
increased heart rate, and lower abdominal
pain.
● It can be accompanied by bacteremia.
5. Ureteritis
● Inflammation or infection within the ureters
(ureteritis) is considered in combination with kidney
infections. UTI within the ureters indicates that
organisms have begun or are in the progress of
ascending into the kidneys and should be treated
similarly to present further infection.
SPECIMEN COLLECTION
CCMS (CLEAN-CATCH MIDSTREAM URINE)
● It is the specimen preferred in patients who can cooperate;
least invasive, preferred routine collection procedure, and
must be performed carefully with optimal _
● Voided midstream collection is the most commonly used
method in clinical practice
● Patients should be educated on how to collection the
specimens
○ guidelines for proper specimen collection should be
prepared on a printed card
○ procedure clearly described and preferably
illustrated to help ensure the patient’s compliance
The instructions on how to collect is very important in order to
isolate the uropathogens
As mentioned on the previous slide, collection of the specimen using
non-invasive techniques may contaminate the sample with normal flora
coming from the urethra
○ That is why you have to educate the patient on how
to collect the specimen
● When giving instructions, include on instructing the
patient to clean the periurethral area
○ include the tip of the penis, labial folds, vulva)
○ carefully clean with 2 separate washes into a plain
soap and water or a mild detergent
○ should be rinsed with warm water to remove the
detergent since some detergents are bacteriostatic
● Instruct the patient to allow the first initial part of the
stream to flush the urethra, and the subsequent
midstream urine is collected on a sterile container to be
used for culture and colony count
Figure 2. Clean-catch Midstream Urine Specimen Collection
STRAIGHT CATHETERIZED URINE 
Specimen collection procedure preferred in patients who cannot
cooperate or in patients who are unable to void because of underlying
physiologic conditions
● This method of collection is slightly more invasive since
urinary catheterization provides a method for the collection of
uncontaminated urine from the bladder, however, there is less
urethral contamination
● Risk: introduction of organism into the bladder with the
catheter
Figure 3. Straight Catheterized Urine Specimen Collection
SUPRAPUBIC BLADDER ASPIRATION
● Like in the straight catheterized urine, this method is usually
done by a physician or another trained health professional to
perform the procedure
● A collection of urine directly into a syringe to a
percutaneously inserted needle during the procedure to
ensure a contamination-free specimen.
● This technique may be indicated in certain clinical situations,
and before collection, the bladder must be full
○ (e.g. done in pediatric practice when urine is difficult
to obtain)
● If a good aseptic technique is used, this procedure can be
performed with a little risk in premature infants, neonates,
small children, pregnant women, and other adults with full
bladders
● Urine is withdrawn directly into a syringe through a
percutaneously inserted needle during suprapubic bladder
aspiration, thereby ensuring a contamination-free specimen.
● Suitable for anaerobic culture since it is free of
contamination from the urethra are collected primarily from
infants and patients in whom interpretation of the results of
voided specs is difficult.
● With the bladder full, the urine is collected with a needle and
syringe following skin antiseptics.
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Figure 4. Suprapubic Bladder Aspiration Urine Specimen Collection
INDWELLING CATHETER
● Usually collected in patients who are housed in the
hospitals and long-term care facilities and those treated in
other healthcare associated settings (e.g. outpatient clinics for
cancer and transplant patients) are more frequently required
to use indwelling urinary catheter.
● These patients are likely to develop bacteriuria which leads to
a more severe infection. This is why it requires a scrupulous
aseptic technique.
● Healthcare workers who manipulate urinary catheters in any
way should wear gloves.
● As seen in the picture below, the catheter tubing should be
clamped above the port to allow collection of freshly voided
urine.
● This catheter port or wall of the tubing should be cleansed
vigorously with 70% ethanol.
● Urine is aspirated using the needle and syringe. The integrity
of the closed drainage system must be maintained to prevent
the introduction of the organisms into the bladder.
● Specimens obtained from the collection bag are inappropriate
or not recommended because organisms can multiply
obscuring the true relative numbers.
● Cultures must be obtained when patients are ill and
routine monitoring does not yield clinically relevant data.
Figure 5. Insertion of Catheter
SPECIMEN TRANSPORT
URINE
● When urine specimens are submitted in the lab for
microbiologic examination, it should be processed right away.
● Processed in the lab within 2 hours, if not, refrigerate
● Excellent supportive medium for growth of most bacteria,
must be refrigerated immediately or preserved
● Bacterial counts in refrigerated urine remain constant for as
long as 24 hours
Figure 6. Urine Transport Kits
● Preservative: urine transport tube (BD urine culture kit)
containing boric acid, glycerol, and sodium formate
○ Preserves bacteria without refrigeration for 48 hours
when more than 100, 000 organisms/mL are present
in the initial urine specimens.
○ must be used with a minimum of 3-5 ml urine to
avoid dilution effect
○ If more than 5 is added, the system will prevent the
growth of certain organisms
○ No advantage over refrigeration except perhaps
convenience
● Another preservative transport: manufacture by Starplex
Scientific, Inc., Cleveland, TN
○ Uses a 10mL sterile conical vial containing a boric
acid tablet that maintains organism viability for up
to 72 hours
○ Both boric acid products preserve bacterial viability
in urine in the absence of antibiotics
○ For patients from whom colony counts of organisms
of less than 100,000 per mL might be clinically
significant within 2 hours of collection is
recommended
○ Convenient method for preserving and transporting
urine from remote areas where refrigeration is not
practical
○
MICROSCOPIC EXAMINATION (SCREENING TEST)
Importance of screening test:
● Screening tests have been developed to quickly identify
those urine specimens that would be negative on culture.
● As a MT, we have to provide the clinician a reliable
screening test for the presence or absence of bacteriuria,
because for the clinician, it can help them guide the therapy of
the patient prior to the availability of the microbiologic data.
GRAM STAINING
● The most commonly used microscopic exam, part of routine
culture
● Differential gram stain is considered the easiest and least
expensive, and probably the most sensitive method for
identifying urine specimens that contain more than 100,000
CFU/ml of urine
● Once a urine specimen is received in the lab, a smear is
made right away and gram staining is performed because the
doctor will call the lab and ask for a preliminary report of the
microscopic results or morphology of the bacteria.
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● In making a smear from urine, the first thing to do is to mix
the urine sample by inverting or mixing the bottle with the
specimen.
● After, using a wire or a calibrated loop, put a drop of the well
mixed urine on the glass slide. Just put a drop, do NOT
spread the urine.
● Air Dry → heat fix and stain → examine under OIO
● Presence of 1-5 organisms per field when examining at
least 20 fields is an indication that the patient has urinary
tract infection, or it correlates with a significant bacteriuria.
● If you do colony count, 1-5 organisms/field is an indication
that the colony count is more than 100,000 CFU/ml urine.
● Picture shows a gram stain smear from the uncentrifuged,
well mixed urine, showing the presence of the pus cells and
the bacteria.
● Take Note: the gram stain should not be relied on for
detecting polymorphonuclear leukocytes in urine because
leukocytes deteriorate quickly in urine that is not fresh or not
adequately preserved.
● Another drawback of the gram stain as a screening test is that
it is not reliable in detecting lower yet clinically significant
members of organisms and because of its labor intensity
● In the lab, if gram stain is used, it should be limited only to
patients with acute pyelonephritis, patients with invasive
urinary tract infections, or other patients for whom
immediate information is necessary for appropriate clinical
management.
circumstances . Why? Because the leukocyte esterase test is not
sensitive enough for determining pyuria in patients with acute
Figure 7. Gram stain smear of uncentrifuged urine
ENUMERATING PMNs IN UNCENTRIFUGED URINE SPECIMENS
● Correlates fairly well with the number of PMNs excreted into
the urine/hr
● Best indicator of the state of the host
● Presence of PMNs is associated with pyuria
● To detect presence of these leukocytes or the PMNs in
uncentrifuged urine, you need to use a hemocytometer. So
you need to perform this using a hemocytometer
● Presence of 10 leukocytes/mm3 is the hallmark of the
inflammation.
● This method of screening urine correlates well with the
number of PMNs excreted per hour which is the best indicator
of the host state.
● Patients with >300.000 PMNs excreted into the urine per
hour are likely to be infected
● We mentioned above that presence of leukocytes or the
PMNs is associated with pyuria, however, this screening test
is not reliable in the detection of pyuria. This is because
pyuria can also be associated with other clinical diseases
such as vaginitis. Therefore, it is not specific for urinary tract
infections
CHEMICAL METHODS
● Another method which is one of the screening tests to detect
urinary tract infection.
● Uses an impregnated filter paper strips
INDIRECT INDICES
● A screening test commonly used to detect bacteriuria and
pyuria by examining for the presence of bacterial enzymes
and/or PMNs enzymes rather than the organisms/PMNs
themselves
NITRATE REDUCTASE (GREISS TEST)
● This screening procedure looks for the presence of urinary
nitrite, an indicator of UTI
● Nitrate-reducing enzymes that are produced by the most
common urinary tract pathogens reduce nitrate to nitrite
● This test has been incorporated into a urinary dipstick that
also test the leukocyte esterase and enzyme produced by the
PMNs
● This test has been impregnated onto a paper strip that also
tests for leukocyte esterase which is an enzyme produced by
PMNs
LEUKOCYTE ESTERASE TEST
● The presence of the PMNs in the urine is an evidence of the
host's response to infection because inflammatory cells
produce leukocyte esterase
● This method can also be just like the nitrate reductase test, it
can be incorporated into the dipstick method
● Simple, inexpensive and rapid method that measures
leukocyte esterase produced by the inflammatory cells
NOTE: this test cannot be used as a stand alone test in most
urethral syndrome
CATALASE : UPI URISCREEN
Figure 8. UPI Uriscreen
● Another rapid urine screening system based on the detection
of catalase present in somatic cells and in most bacterial
species commonly causing UTI’s except streptococci and
enterococci
● We call this screening test as the Catalase or API uriscreen or
the accutest uriscreen
● How to perform this catalase:
○ Substrate : add 1.5-2.0 ml urine -> mix
○ (+) result: formation of the bubbles above the
liquid surface
Figure 9. Positive result
○ Approximately, 1.5-2 ml of urine -> then you add this
to the tube containing dehydrated substrate -> then
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after which , you add hydrogen peroxide to the urine
and you mix gently
AUTOMATED & SEMI AUTOMATED SYSTEM
So aside from the conventional methods used to detect renal tract
infections , we can now also use automated and semi automated
system
Figure 10. Automated & Semi automated system instrument
● Advantage of this system over conventional methods
○ Minimal labor and rapid turnaround time
compared to conventional cultures
Examples of the automated system:
4.1 YELLOW IRIS SYSTEM :
(INTERNATIONAL REMOTE IMAGING SYSTEM)
● So this system is capable of analyzing the urine or body
fluid sample in 1 instrument. So the instrument analyzes
both the microscopic components and the urine
chemistry by combining technology of both types of
analyzers into 1 automated system
● This iris system uses a Flow Imagine microscopy method
to capture individual images to each particle identified in the
specimen then classifies the particle .
● So meaning: this system is Is able to recognize many cellular
structures, including the leukocytes and bacteria by examining
As mentioned previously, urine samples may be contaminated with the
the images of uncentrifuged urine samples using video
presence of normal flora coming from the distal portion of the urethra,
camera
especially if the urine is collected with a non-invasive technique. Hence,
● (we have this instrument in the CVGH lab)
a quantitative culture is done in the laboratory to differentiate the
colonization, infection, and contamination.
4.2 PHOTOMETRY : (VITEK SYSTEM)
Figure 11. Vitek System
● This is another system used to detect urinary tract infections
● This method uses photometry for the physical and
chemical identification of organisms and other particles
in the urine samples
● If a significant number of organisms are present in the urine
specimen, they will grow in the medium to a detectable conc.
using photometry
● So it can detect a colony count of between 10,000 to 100 000
CFU / ml
○ > 10 4 -10 5 CFU/ml
4.3 COLORIMETRIC PARTICLE: FILTRATION
(BAC-T-SCREEN 2000)
Figure 12. Bac-T-screen 2000
● Are used to trap organisms and WBC’s on filters and then
selectively stains the cells
● Uses the : (Safranin O dye)
● So just like the Vitek system, this type of machine can detect
a colony count of between 10 000 to 100 000 CFU/ml
○ > 10 4 - 10 5 CFU/ ml
DRAWBACK OF SCREENING PROCEDURES
● Screening methods are insensitive ar levels below 10 5 CFU/
ml
● Therefore they are not acceptable for urine specimens
collected by suprapubic aspiration, catheterization or
cystoscopy
● Screening methods may also fail to detect a significant
number of infections in symptomatic patients with low colony
counts (100-1000 CFU/ml) such as young, sexually active
females with acute urethral syndrome.
METHODS OF CULTURING URINE AND INTERPRETATIVE
CRITERIA
1. “CALIBRATED LOOP DIRECT STREAK METHOD”
● A calibrated loop is used to do a quantitative count of the
colonies growing on the plate
● ROUTINE CULTURE MEDIA USED
● Primary inoculation: With the calibrated loop, one loop full of
urine specimen is inoculated into the blood agar
● 5% Sheep Blood Agar or MacConkey Agar or EMB
○ Allows detection of most gram (-) bacilli,
staphylococci, streptococci, and enterococci
● SELECTIVE MEDIA MAY BE USED
● (e.g. enterococci and other streptococci may be obscured by
the heavy growth Enterobacter spp.) for the isolation of gram
(+) organisms:
○ CNA (Columbia-Colistin-Nalidixic acid) Agar
○ PEA (Phenylethyl Alcohol Agar)
○ Enterococcosel Agar (Bile Esculin Azide Agar)
○ Cystine-lactose-electrolyte deficient (CLED)
■ support the growth of most common
urinary pathogens and inhibits the
swarming growth of Proteus spp.
○ BD CHROMagar
■ Uses enzymatic reactions to identify E.coli
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■ Allows more specific direct detection and
differentiation of urinary pathogens on
primary plating.
INOCULATION AND INCUBATION OF URINE CULTURES
INOCULATION INSTRUMENT:
● Calibrated wire inoculating loop: 0.01ml or 0.001ml
● These loops, made of platinum, plastic, or other material, can
be obtained from laboratory supply companies
● The calibrated loop that delivers the larger volume of urine
(0.01ml) is recommended to detect lower numbers of
organisms in certain specimens (e.g. specimens collected
with invasive techniques).
○ For example, urine collected from catheterization,
nephrostomiesileal conduits, and suprapubic
aspirates should be plated with a larger calibrated
loop.
PROCEDURE:
1. Flame the calibrated loop and allow it to cool without touching
any surface. (Aseptic Technique)
2. Mix urine thoroughly and remove the top of the container.
3. Insert loop vertically into the urine to allow it to adhere to the
loop.
4. Spread the loopful of urine over the surface of the agar plate
as illustrated.
4.1. Loop is touched to the center of the plate, then spread
the inoculum in a line across the diameter of the plate.
4.2. Without flaming or reentering urine, loop is drawn across
the entire plate, crossing the first inoculum streak numerous
times to produce isolated colonies.
5. Without reflaming, insert the loop vertically into the urine again
for transfer of a loopful to a second plate. Repeat for each
plate.
6. Incubate the plates for at least 24 hours at 35-37°C in air.
➢ A minimum of 24 hours is typically necessary to detect
uropathogens.
➢ Some specimens inoculated late in the day cannot be
read accurately the next morning, so these cultures are
re-incubated the next day or interpreted later in a day
when a full 24 hours incubation has been completed.
Colonies are counted on each plate. The number of CFU’s is
multiplied by 1000 (if a 0.001 ml loop was used) or by 100 (if
a 0.01 ml loop was used) to determine the number of
microorganisms/ml of the original specimen.
● In the CVGH Lab, they do the colony count using
the BA plate.
7. Because antimicrobial treatment or other factors may inhibit
initial growth, reincubate plates with no growth or tiny colonies
for an additional 24 hours before discarding plates.
8. To store the inoculating loop, place (handle down) in a test
tube taped to the wall, rather flat on the bench, to prevent
bending, which would destroy the calibration.
Classical Method of Interpreting Urine Culture:
● Popularized by Edward Kass
Figure 13. Label
● >100,000 CFU/mL = significant (top)
● 10,000 - 100,000 CFU/mL = possible infection (top)
● <10,000 CFU/mL = contamination (bottom)
○ Contamination may probably come from the urethra,
since it contains a large number of normal flora.
That is why we do quantitative culture in order to
discriminate between colonization, infection, and
contamination.
● One of the major problems in interpreting urine cultures arises
because of urine samples by a voided technique or
non-invasive technique, in which the sample may be
contaminated with the normal microbiota including the
Enterobactericiae.
○ Determining what colony count represents true
infection from contamination is of utmost importance
and is related to the patient's clinical presentation.
● Increasing difficulty in distinguishing between infection and
contamination as a criteria for positive culture: The colony
count is lowered from 100,000 CFU/mL to 100 CFU/mL
○ Because of the variation of the interpretative
guidelines occurring from one laboratory to another,
many laboratories established their own
interpretative criteria for urine cultures based on the
type of specimen submitted.
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MICRO 1: TITLE OF LESSON
GENERAL INTERPRETATIVE GUIDELINES FOR URINE CULTURE:
104 (OR 10,000) CFU/ML OF A SINGLE POTENTIAL PATHOGEN OR
FOR EACH OF TWO POTENTIAL PATHOGENS
● (CCMS, pyelonephritis, acute cystitis, asymptomatic
bacteriuria, catheterized urine)
WORK UP IS COMPLETE
● (ID and susceptibility testing is done on the specimen)
103 (OR 1,000) CFU/ML OF A SINGLE PATHOGEN
● (CCMS urine or symptomatic males or catheterized urine or
acute urethral urine)
WORK UP IS COMPLETE
● (ID and susceptibility testing is done on the specimen)
>3 ORGANISM TYPES WITH NO PREDOMINATING ORGANISMS
● (CCMS urine or catheterized urines)
WORK UP: NONE (POSSIBLE CONTAMINATION);
● Ask for another specimen
● Either 2 or 3 organism types with predominant growth of 1
organism type (CCMS urine)
● Complete work-up of predominating organisms: 104 - 105
CFU/mL
>102 CFU/ML OF ANY NUMBER OF ORGANISM TYPES IN A
SET-UP WITH A 0.001 AND 0.01 ML CALIBRATED LOOP
○ Specimen from suprapubic aspirates, or any other
surgically obtained urines (including ileal consults,
cystoscopy specimens)
○ Workup is complete
○ This is considered significant because the
specimens are collected using invasive techniques
which is free of urethral contamination
○ Do identification and appropriate susceptibility
testing
● A pure culture of S. aureus is significant regardless of the
number of CFU’s and susceptibility tests are performed
● The presence of yeasts in any number is reported to the
physician
○ Because the presence of these uropathogens is an
indication that patient has pyelonephritis
Figure 14. Label
● Culture results illustrating some of the various interpretive
guidelines
A [MACCONKEY MEDIUM]:
● You can see the growth or colonies of ≥105 CFU/mL of a
lactose-fermenting gram-negative rod in a clean-catch
midstream (CCMS) urine from a patient with pyelonephritis; a
complete workup would be done
B:
● Growth of ≥105 CFU/mL of a lactose-fermenting
gram-negative rod (arrow A) and <104 CFU/mL of another
organism type (arrow B) from a CCMS urine; only the
organism with a colony count of >104 - 105 CFU/mL would be
worked up completely
Figure 15. Label
● For primary inoculation, we inoculated BA and MacConkey
and made a smear from the specimen. After inoculation, the
plate is incubated for 18-24 hrs at 37°C.
● If your smear will show Gram (-) rods, you have to examine
your MacConkey plate after incubation for the presence of
growth
● If Gram (+) Staphylococci are seen in the Gram stain smear,
you have to examine for the presence of typical colonies of
Staphylococci that will grow on blood agar
● Once white opaque beta-hemolytic colonies are seen on the
blood agar plate, make a smear from the culture (isolated
colonies on the blood agar plate)
○ If it shows Gram (+) Staphylococci, proceed to
identification
○ To differentiate S. aureus from S. epidermidis, do
Slide coagulase
○ If slide coagulase is (+), the isolate is S. aureus
○ But if ever your slide coagulase is negative, you
proceed to tube coagulase.
○ If your tube coagulase is negative, your isolate is
staphylococcus epidermidis.
WHAT IF GRAM NEGATIVE RODS ARE SEEN ON YOUR
MCCONKEY?
○ After 24 hours of incubation on McConkey, there are pink
colored colonies growing on McConkey, next step is to make
a smear from the culture, meaning, form the colonies growing
on McConkey.
○ If the smear from your culture will show Gram negative (-)
rods, you proceed to biochemical test and identification.
GENITAL TRACT INFECTIONS
● General tract infections (GTI’s) and sexually transmitted
infections (STI’s) are caused by organisms normally present
in the reproductive tract, or introduced from outside during
medical procedures or during sexual contact.
BACTERIAL FLORA OF GENITAL TRACT:
● The lining of the normal human genital tract is a mucosal layer
made up of transitional columnar and squamous epithelial
cells.
● Various species of commensal bacteria colonized these
surfaces, causing no harm to the host except under abnormal
circumstances.
● Thus, presence of this bacteria will help the host prevent the
adherence of pathogenic organisms.
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MICRO 1: TITLE OF LESSON

● In the female genital tract, the mucosal surfaces are normally ● Other yeasts
colonized with several species of bacteria which are usually
considered as normal and even protect them from the VIRUSES
adherence of the pathogenic organisms.
● Cytomegalovirus
WHAT ARE THE NORMAL URETHRAL FLORA?
● HIV
○ The distal portion of the urethra is colonized by large number ● HSV
of normal flora.
○ This normal urethral flora can also be part of the normal flora PROTOZOANS (STD’S)
of the human genital tract like your coagulase negative (-)
staphylococci, corynebacteria, and a number of anaerobes. ● T. vaginalis
THE VULVA AND THE PENIS
GIT AND SYSTEMIC PATHOGENS AS STD AGENTS
● especially the area underneath the pubic of uncircumcised GIT PATHOGENS
male is a mixture of organisms present on the skin of this
area: ● G. lamblia
○ Mycobacterium smegmatis (M. smegmatis) ● E. histolytica
○ Other gram(+) bacteria ● Cryptosporidium
● Microsporidium
THE FLORA OF THE FEMALE GENITAL TRACT VARIES WITH:

● pH SYSTEMIC PATHOGENS
● Estrogen concentration of the mucosa (which depends on the
host’s age) ● Salmonella
● Shigella
PREPUBESCENT & POSTMENOPAUSAL WOMEN: ● Campylobacter

● Harbor primarily staphylococci & corynebacterium Route of infection: anal genital route (common in homosexual or
heterosexual practices)
WOMEN OF REPRODUCTIVE AGE:
LABORATORY DIAGNOSIS OF GIT’S: LOWER GIT (URETHRITIS,
● May harbor large numbers of facultative bacteria:
CERVICITIS AND VAGINITIS)
○ Enterobacteriaceae
○ Streptococci
○ Staphylococci ● in the laboratory, we usually collect genital tract specimens to
determine the causative agents of urethritis, cervicitis, and
ANAEROBES: vaginitis as well as childbirth infections
LACTOBACILLI ● most often, genital tract specimens are collected to determine
the presence of N. gonorrheae (agent of gonorrhea) and C.
■ predominant organisms in secretions from normal healthy trachomatis (common cause of cervicitis)
vaginas ● specimens collected from females with suspected genital
- Deoderlein bacillus,
infections are most frequently from the uterine cervix and the
- Anaerobic nonsporeforming bacilli & cocci
- Clostridia urethra

YEASTS
- acquired from GIT; May be transiently recovered from female
vaginal tract, but are not normal flora.
ORGANISMS RECOVERED FROM THE GENITAL TRACT THAT
ARE USUALLY CONSIDERED TO BE PATHOGENS
(are the etiologic agent for STD’s and genital tract infections):
BACTERIA
SPECIMEN COLLECTION: URETHRAL DISCHARGE
● most common specimen collected in order to diagnose
genital tract infections
● may occur in both males & females infected with N.
gonorrhoeae & T. vaginalis
● less profuse in female & may be masked by vaginal discharge
● recommended only for male patients
● U. urealyticum can also be isolated from male urethral
discharge

● C. trachomatis
● M. hominis
● U.urealyticum
● M. genitalium
● G. vaginalis
● T. pallidum
● N. gonorrhoeae (common in PH)

FUNGI

● Candida spp.
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MICRO 1: TITLE OF LESSON

Figure 16. Label

UROGENITAL SWAB

● ideally collected 1 hour after urination

● Thin swab made of cotton or rayon that has been treated with
charcoal to adsorb material toxic to gonococci
● We usually do bedside inoculation
○ so we bring the plate or respective medium for the
isolation of N.gonorrhoeae when you go into the
ward and you have to do it quickly because
N.gonorrhoeae is susceptible to temperature
fluctuations such as cooling and does not sustain
drying or low levels of carbon dioxide; thats why its
very important to do bedside and you make a smear
Figure 18. Label
then transport quickly
● Same organism causing purulent infection in urethra.
PROCEDURE ● Procedure:
○ usually the gynecologist or the obstetrician who will
1. Insert approximately 2cm into the urethra & rotate gently perform the procedure.
before before withdrawing to remove epithelial cells from ○ Mucous is removed by gently rubbing the area with
urethra mucosa (Chlamydia - intracellular) a cotton ball.
● Esp. when you want to isolate chlamydia which is an ○ Insert the urethral swab into the cervical canal,
intracellular organism rotate and move side to side for 30 seconds before
2. Or you can Separate sample for cultivation of GC removal.
(gonococci), chlamydia & ureaplasma ○ Endocervical specimens:
■ Collected using speculum.
● In the CVGH lab you can, instead of using a urogenital swab ■ Avoid contaminating swab with vaginal
we can use an inoculating wire , it's more sensitive than using secretions.
the swab
○ So when you go to the ward you need to bring SWABS COLLECTED FOR ISOLATION OF GONOCOCCI
alcohol lamp because you need to do aseptic
technique before inserting the loop
● Neisseria gonorrhoeae is very susceptible to temperature
○ Before collection you have to explain to the patient
fluctuations (e.g. cooling) and does not sustain drying.
that you are going to insert the loop and why you
● It is very important to smear, plate, and transport the
need to heat the loop before you insert into the
specimen quickly.
urethra because makuyawan nya ang patient
● have to inoculate the specimen into a selective medium (e.g.
masumbagan mo
Thayer Martin Medium)
○ Or ma rattle mo then iinsert ninyo while it's still hot
● if you cannot inoculate the specimen right away, it may be
ma paso ma double dead ang patient, you didnt
transported in modified Stuart’s or Amies Charcoal TM held at
orient then ma paso pa jd so explain it to the patient
RT until inoculated to culture media
○ specimens collected which are suspected or contain
gonococci should not be refrigerated, otherwise,
they will be killed, and you cannot isolate the
organisms anymore
● culture within 12 hours of collection
○ >12 hours: should be inoculated directly to one of
commercial systems (Jembec plate/ Transgrow
bottle)
Figure 17. Label
SWABS FOR ISOLATION OF CHLAMYDIA/MYCOPLASMA ARE
BEST TRANSPORTED
● In cases where there is profuse urethral discharge, (pic
below) particularly in males, the discharge maybe be collected ● should be transported in specific TM with antibiotics
externally without inserting the device into the urethra ● specimens for Chlamydia culture should be transported on ice
● Another one is you can use a Few drops of first-voided urine ○ if you cannot transport it on ice, specimen
specimen can detect gonococci in males transported at RT should be inoculated within 15
minutes of collection
● Specimen transported at RT should be inoculated within 15
FEMALES
minutes of collection
● Medtechs do not collect specimen from females it's the
gynecologist or the obstetrician so usually doctors that DIRECT MICROSCOPIC EXAMINATION
collects this
❖ Gram staining
CERVICAL/VAGINAL
➢ Most commonly used direct microscopic
● Organism that cause purulent vaginal discharge (vaginitis) examination to visualize the microscopic
include: morphology of the organism
○ T.vaginalis, gonococci & rarely, beta-hemolytic
streptococci ● Gram (-) intracellular diplococci in urethral discharge:
■ Same organism causing purulent infection usually indicative of gonococci (GC) (gonorrhea) in males
in urethra

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MICRO 1: TITLE OF LESSON

○ do bedside inoculation. ○ When doing wet mount, examine the characteristic

○ after inoculation to culture media, the swab is rolled motility of the parasite
over the surface of the glass slide covering an area ● Stain smear with the I and H to see the characteristic
of at least 1 sq.cm trophozoite of T. vaginalis
○ as seen on the image, there are numerous
polymorphonuclear neutrophils which are usually
>4/OIO field
■ an indication that the px has an acute
urethritis.
○ If the gram stain is diagnostic, cultures of urethral
discharge may not be performed.

Figure 21. T. vaginalis as seen microscopically

BACTERIAL VAGINOSIS

● third type of vaginitis

● characterized by foul smelling discharge, can be diagnosed
microscopically/clinically
● discharge is primarily sloughed epithelial cells covered by tiny
Figure 19. Numerous polymorphomuclear neutrophils as seen Gram variable rods & coccobacilli (clue cells: an indication of
microscopically bacterial vaginosis)

● In females: R/O Veillonella, gram (-) coccobacilli may

resemble gonococci.

○ Presumptive diagnosis for gonorrhea from these

smears is only reliable if the
microscopist/microbiologist/MT is experienced
because normal flora such as the R/O Veillonella,
gram (-) coccobacilli may resemble gonococci
○ Culture should always be performed on specimens
from females. Figure 22. Epithelial cells from swabbed discharge with clue cells

FLOURESCIN-CONJUGATED MONOCLONAL AB REAGENTS DIAGNOSING BACTERIAL VAGINOSIS CLINICALLY

A clinical diagnosis of BV is best made using three or more of the

following criteria:
1. Macroscopic examination of discharge: Homogenous, gray
discharge
2. Presence of clue cells on a wet mount or gram stain
3. An amine or fishy odor elicited by the addition of a drop of
10% KOH to the discharge on a site or on the speculum
4. pH > 4.5
5. Absence of inflammatory cells in the vaginal discharge is
another sign
a. Although G. vaginalis has been historically
associated with the syndrome and can be cultured
on human blood bilayer plate, however, culture is
not recommended for the diagnosis of bacterial
vaginosis
● best way to differentiate bacterial vaginosis from other vaginal
discharge infection is through Gram stain
Figure 20. C. trachomatis visualization (?)
○ Grading system is based in the presence or
absence of certain bacterial morphologies
● Presumptive identification of the presence of inclusion
bodies of C. trachomatis
● Sensitive and specific for visualization of inclusion bodies in
cell cultures and elementary bodies in urethral & cervical
specimen containing cells

EXAMINATION OF WET PREPARATION OF VAGINAL DISCHARGE

● Rapid diagnostic test for T. vaginalis (one of the etiologic

agents of STD)
● To diagnose Trichomonas, we can do a wet mount
preparation of the vaginal discharge.
○ Once received in the lab, perform test right away
because these organisms are motile trophozoites
which die rapidly

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MICRO 1: TITLE OF LESSON

Figure 23. In patients with BV, lactobacilli are either absent or few in ● When you inoculate the specimen, you have to inoculate the
number while curved, Gram(v) rods Mobiluncus and/or G. vaginalis and swab first into the plated medium.
Bacteroides morphotypes predominates. ● For primary inoculation, we inoculated in BA, CA, TH, THIO,
and MC agar.
PROCESSING URETHRAL SPECIMENS SUSPECTED TO CONTAIN ● When you inoculate the specimen, you have to roll all areas of
GONOCOCCI the swab into the inoculum well in order to expose all surfaces
onto the medium. Then, you proceed with four-quadrant
CULTURE streaking.
● After inoculation, all plates should be incubated at 37°C for
18-24 hours.

BA CA TM THIO MC

incubate in candle jar or incubated

under increased CO2 aerobically
tension
Figure 24. Label

● samples for isolation of GC may be inoculated directly to
culture media (do a bedside inoculation since N. gonorrhoea
is very susceptible to temperature fluctuations (e.g. cooling)
and does not sustain drying in low levels of CO2)
● culture media:
○ Mod. Thayer-Martin is most often used
○ New York City (NYC) medium has the added
advantage of supporting the growth of mycoplasmas
and GC
bilat
otin
● When you examine the gram stain smear and you see the
presence of gram (-) diplococci, after incubation, you have to
examine your CA and TM for the presence of growth.
○ If grayish, white, moist, translucent colonies are
seen growing on your CA or TM, the next step is you
make a smear from the CA or TM culture to confirm
the presence of gram (-) diplococci.
● If a gram (-) diplococci is seen from the smear, you proceed
with identification.

SELF-CONTAINED INCUBATION SYSTEMS IDENTIFYING NEISSERIA

● First, you have to do an oxidase test (presumptive test) to

identify the species of Neisseria.
● To identify the species, we do a confirmatory test in which
we perform the carbohydrate fermentation test.
● N. gonorrhoeae will produce acid in glucose only, all other
sugars are negative.
○ This means that only glucose is fermented. Your
isolate now is N. gonorrhoeae.

Figure 25. Jembec Plate (Right), left

● if immediate inoculation or bedside inoculation is not possible,

the specimen should be placed in a transport media and sent
to the lab or you can also inoculate the specimen directly to
the self-contained incubation system
○ Jembec Plates which generates its own increased
CO2 atmosphere by means of sodium carbonate
tablet
■ plate should be inoculated in W-pattern

Figure 26. schematic diagram showing the isolation and identification

of Neisseria gonorrhoeae from urethral discharge

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