MICROBIOLOGY LABORATORY

By : Edessa S. Valenzuela, RMT.

COLONY COUNT
 Significance of Colony Counts:
• Laboratory Diagnosis
Historical Background
The urinary tract above the urethra in normal healthy individuals
is sterile, as is urine, unlike the urethra, which is colonized with
perineal flora. In women, vaginal flora may contaminate the urine;
many of the organisms that colonize the vagina are also implicated
in UTIs. Therefore, it could be difficult to know whether growth in
urine cultures was indicative of infection or contamination.
Since 1956, the interpretation of quantitative urine cultures
has been considered one of the more straightforward and
simpler
laboratory tests to diagnose UTIs. It was understood that a
finding
of 100,000 (105) CFU/mL or more was a positive test result,
symbolizing infection. In his 1956 classic study, Kass showed a
clear separation between the number of bacteria in the urine of
asymptomatic or symptomatic women with pyelonephritis and
those who were uninfected. Significantly, 95% had colony counts
higher than 105 CFU/mL when infected
• In retrospect, this study had some definitive parameters— namely, that all
specimens were collected by catheterization (not voided), most
asymptomatic women had counts below 103 CFU/
mL, and the prevalence of infection was only 6%. With this low prevalence
rate, the number of false-positives would be higher, hence the 105 CFU/mL
so-called positive infection cutoff point.

• In 1980, the acute urethral syndrome, or urethritis, was more clearly defined
as one of the three causes of acute dysuria with pyuria. The other
causes were vaginitis and cystitis. The causative organisms included classic
coliforms and Staphylococcus saprophyticus at colony counts above 103 but
below 105 CFU/mL. Simultaneously implicated in sexually active women were
the emerging nongonococcal urethritis pathogens, Chlamydia trachomatis
and Ureaplasma urealyticum. Thus, in women with UTIs, as many as 50%
had urethral syndrome—not cystitis
• Until recently, the colony count was regarded as the gold standard for
determining whether a patient had a real and treatable UTI; however, the
urine bacterial colony count cannot stand alone as a single criterion when
evaluating for the presence or absence of UTI. Urine cultures are requested
not only in connection with acute UTI symptoms but also in the absence of
specific symptoms, as a test of cure, to evaluate antimicrobial therapy
effectiveness, detect ASB in pregnant women, and evaluate for
bacteremia, fever, or both, to name a few. Also, the criteria that
determine whether a UTI is present must include the presence or
absence of symptoms, predisposing factors, patient population,
and type of organism(s) isolated. The outcome of a urine culture
therefore must be evaluated along with other laboratory and clinical data;
attempts to attach significance to the colony count should be restricted to
the original patient population in which that significance was established,
asymptomatic individuals with pyelonephritis.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
 BACKGROUND
Most in vitro antibiotic susceptibility testing in clinical laboratories
is still performed with the use of phenotypic methods, which are
simple and inexpensive. Phenotypic methods require isolation of
the
pathogen being tested, followed by exposure of the pathogen to
the antimicrobial and subsequent evaluation of the expression, or
lack of expression, of resistance to the antibiotic.
 DEFINITION
• Antimicrobial susceptibility tests may be categorized according to the
endpoint used, that is, inhibition of growth or killing. In most cases, inhibition of
growth is the parameter used in the laboratory, with only limited use of a killing
endpoint for very specific circumstances
The inhibitory parameter that forms the basis for the majority of phenotypic
susceptibility tests is the MIC, which is the lowest concentration
of antibiotic that inhibits the visible growth of an organism in an in vitro
system. MIC results are dependent not only on the interaction between the
antimicrobial agent and the organism, but also on test conditions.

These conditions include pH and ion concentrations of testing media, the

temperature at which the test system is incubated, the incubation atmosphere, the
amount of organism used in testing, and the length of time the system incubates. To
allow intralaboratory and interlaboratory reproducibility, these test conditions have
been standardized. Various standards organizations in a variety of venues throughout
the world have set and published these parameters, and in the United States, the CLSI
fulfils this role.
 Areas of Standardization for
Susceptibility Testing
• Growth medium
• pH (7.2 – 7.4)
• Atmosphere (ambient air, no carbon dioxide)
• Inoculum (McFarland 0.5 standard)
• Antibiotics (stored properly)
• Quality control
 INTERPRETATION of RESULTS
• The basic interpretative categories include susceptible, intermediate, and
resistant.
1. Susceptible implies that therapy with the recommended dosage of a particular
antibiotic is likely to be effective in eradicating the infection
2. Resistant indicates that the antibiotic in the appropriate dose has not been
shown to have a high likelihood of treatment success in clinical trials.
3. Intermediate suggests that the isolate tested may be less inhibited by the usual
dose of the antibiotic than those isolates that are categorized as susceptible,
and that therapy with higher doses of antibiotic or therapy with the
antibiotic for infection in anatomic locations in which the antibiotic normally is
concentrated is likely to be effective.
Heavy inoculum

Antibiotic disk with vernier caliper

 KIRBY-BAUER OR DISK DIFFUSION METHOD
• Measures the diameter of inhibition around the antibiotic-impregnated
filter paper disk. As soon as the disk comes in contact with the agar
surface, water is absorbed into the filter paper and the antibiotic diffuses
into the surrounding medium. The concentration of antibiotic decreases
with increased distance from the disk. If the test is properly performed.
The edges of the zone of inhibition are clear and easy to measure. There
are times when it is not obvious.
1. Swarming (Proteus) – the outside diameter should be measured.
2. Mutants/ mixed culture – colonies that grow in areas of inhibition; this
means that the organism is resistant
3. Overlapping – this makes the result inaccurate.
4. Control plates- performed weekly using stock cultures.
MH agar with swarming motility
MH agar with overlappping of inhibition