Download Diaslide Urine Culture Device

Urine Culture Device
Support Book
April 2002
Confidential
Table of Contents
Diaslide - A Unique Urine Culture Device ................................... 3
Urinary Tract Infections (UTI) - An Overview .............................. 4
General Background .................................................................. 4
Urinary Pathogens ..................................................................... 5
Laboratory Diagnosis ................................................................. 9
Sample Collection ...................................................................... 9
Transportation of urine specimens to the laboratory .................. 9
Culturing microorganisms......................................................... 10
Diaslide® - Unique Product...................................................... 12
Diaslide® Design ...................................................................... 12
Diaslide Packaging ................................................................. 13
Correct storage ........................................................................ 13
Shelf life ................................................................................... 14
Precautions .............................................................................. 14
Diaslide® Procedure ................................................................. 14
Reading the Results ................................................................. 16
Diaslide® vs. Culture media ...................................................... 19
Diaslide® vs. dipslide ................................................................ 20
Who are your Customers? ....................................................... 22
Competition ............................................................................ 23
Summary of competition........................................................... 23
Competitive Analysis ................................................................ 24
List of Competitors ................................................................... 25
Troubleshooting ...................................................................... 29
Published Articles .................................................................... 31
Package Inserts ....................................................................... 32
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Diaslide - A Unique Urine
Culture Device
Diaslide® is a semi quantitative culture method for
the detection of urinary tract infections
demonstrating bacterial counts and isolated
colonies.
Diaslide® consist of a hinged plastic case
containing two opposing agar media (CLED and
MacConkey), separated by a sampler with a
handle - at one end, and two bent sampler
prongs at the opposite end.
Operation
 The tips of the sampler are first dipped into
the urine.
 Simple
 The sampler is then pulled out through the
casing, simultaneously inoculating both agar
surfaces with a streaking dilution.
 Convenient
 After 24 hours of vertical incubation
individual colonies can be observed even
when bacterial concentration exceed 10 6
cells/ml. The number of colonies on the
Diaslide® correlate linearly with bacterial
concentration.

3
Effective
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Urinary Tract Infections
(UTI) - An Overview
General Background
Urinary tract infections are among the most
common infections in humans. The majority of
cases are caused by a limited number of
bacterial genera. The presence of these bacteria
in urine is termed bacteriuria.
Significant bacteriuria (105 cells/ml of urine)
defines the number of bacteria in midstream
clean voided urine that exceed the numbers
usually caused by contamination from the
urethra.
Urinary tract infection implies a bacterial attack
on tissue located anywhere from the renal cortex
in the kidney (upper tract infections), to the
bladder (lower tract infections).
It is generally accepted that urinary tract
infections are usually a consequence of
ascending infection by microorganisms from the
lower tract into the upper tract. Reflux of urine
into the urethra and up into the renal pelvis is
probably an important mechanism for introducing
organisms into the upper tract.
Differentiation between lower and upper
infections is difficult.
Most urinary tract infections are asymptomatic.
One third of the patients with asymptomatic
bacteriuria have an upper tract infection.
• Bacteriuria is
termed for the
presence of
bacteria in urine
• Upper tract
infections renal cortex of
the kidney
• Lower tract
infections
bladder
Clinical symptoms include:
 Urgency and/or frequency in urinating
 Fever
 Increased Erythrocyte sedimentation rate
 Urine sediment of Leukocytes, Bacteria and
Erythrocytes
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Epidemiology
Frequency of bacteriuria in neonates is about 1%
and more common in boys than in girls.
Frequency of bacteriuria in adult men is less
than 1%. After age of 65 bacteriuria frequency
increases and reaches 5-10% at the age of 80.
This increase is attributed at least in part to the
development of prostatic hypertrophy and
obstruction of the bladder outlet.
Frequency of bacteriuria in women increases with
the increasing of age.
In schoolgirls infectious rate is about 1%, and in
young women the range is between 1-3% with at
least 10-20% of adult females experiencing a
symptomatic infection at some time during their
life. In elderly woman the frequency increases to
about 20%.
• Neonates:
Low Frequency
of 1%
• Adult men:
Low Frequency
~1%, increases
with age to 5-10%
• Adult women:
Increases with
age
Urinary Pathogens
Urinary pathogen microorganisms have been well
recognized.
The following list includes those known to cause
infection by the ascending route (by the urethra):
Bacteria
Gram-negative bacilli
Enterobateriaceae Escherichia coli
Proteus spp.
Klebsiella spp.
Citrobacter spp.
Serratia spp.
Enterobacter spp.
Providencia spp.
Pseudomonaceae Pseudomonas spp
Alcaligenes spp.
Gram-negative
coccobacilli
Acinetobacter spp.
Gram-Positive cocci
Micrococcaceae Staphylococcus aureus
Staphylococcus saprophyticus
Staphylococcus epidermidis
Streptococcaceae Streptococcus faecalis
Streptococcus agalactiae
Fungi
Candida spp.
Torulopsis glabrata
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The majority of these organisms are facultative
anaerobes - they can grow either in the presence
or absence of molecular oxygen.
Most of them originate in the commensal flora of
the bowel. Some, including Staphylococcus
epidermidis, group B Streptococci, Candida and
Torulopsis probably originate in the flora of the
vagina or perineal skin in females, or the
perpetual sac in males.
Escherichia coli is the most common urinary
pathogen.
Much less common infections outside hospital
are due to proteus spp., Staphylococcus
saprophyticus, Klebsiella spp., Streptococcus
faecalis and Lancefield group-B hemolytic
streptococci.
• Facultative
anaerobes - can
grow either in the
presence or
absence of
molecular
oxygen
• Escherichia coli
is the most
common urinary
pathogen
Note:
GRAM POSITIVE & GRAM NEGATIVE
BACTERIA
A primary separation of bacteria is based on the
nature of the bounding layer of the cell:
Gram Positive, which synthesizes a monolayer cell
wall and Gram negative, which synthesizes a cell wall
composed of at least two structurally distinct layers.
A differential stain of great practical value for this
identification was discovered empirically in 1884 by
Christian Gram:
A heat fixed smear of cells is stained successively
with the primary stain, crystal violet, and fixed with a
mordant decolorize to remove the unbound stain..
Gram Positive resist decolorization and remain
stained with a deep blue- black color., Gram Negative
bacteria are rapidly and completely decolorized.
A counterstain, safranin, is added to provide color to
Gram negative bacteria.
The Gram positive bacteria will be stained a purple
color, and the Gram negative bacteria, a red color.
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Different Pathogens infect different age
and sex groups
While E.Coli is undoubtedly the most common
urinary pathogen over all, it is not surprising, in
view of the anatomical and physiological
differences between males and females, that
there are differences in the infecting organisms in
particular age and sex groups.
In neonatal infection, when the route of infection
may be haematogenous, the most common
infecting organism in both sexes is E.Coli.
During childhood, however, there are marked
differences:
The frequency with which Proteus spp. causes
infection in boys is striking. This is possibly due to
the presence of this pathogen in the perpetual
sac, the urethra, or even the prostatic ducts.
Proteus becomes much less common in adult
males, only to reappear in old age, suggesting
that prostatic function may play a part in
maintaining an environment in the urethra which
is unfavorable to this species.
E.Coli infection occurs throughout all age groups.
Staphylococcus saprophyticus is the second
most common urinary pathogen in young women
between 15 and 35 years of age, and may be
associated with sexual intercourse. It has been
demonstrated in the rectal flora of adult women,
and infection may therefore arise in the same
way as E. coli infections.
Strains of Staphylococcus epidermidis which are
usually commensal in the urethra may colonize in
the bladder, sometimes causing urinary
symptoms and systemic upset, in patients who
are catheterized. These incidents are very
common in elderly patients undergoing urological
procedures, or in patients of both sexes who are
catheterized for relief of obstruction or
incontinence.
Staphylococcus epidermidis may also cause
infection in patients with renal scars or stones,
who have been subjected to repeated courses of
antibacterial treatment.
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• Anatomical &
physiological
differences
between genders
result in different
infecting
organisms
• The most
common
infecting
organism in
neonates is
E.Coli
• Proteus spp.
causes most
infections in
boys and old
men
• Staphylococcus
saprophyticus is
the second
common
pathogen in
young sexually
active women
• Staphylococcus
epidermidis
infection is
typical to
catheterized
patients
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In Summary:
Different pathogens infect different age
and sex groups, as a result of a
combination of factors:
 Anatomical
differences
 The comensal flora of the anatomical areas
from which ascending infection may occur.
 Antibacterial
factors as
hormones
 The presence of antibacterial factors in, for
instance, prostatic fluid, or produced by
uroepithelium and probably influences of
different sexual hormones.
 Mechanical
interference
 Mechanical factors such as catheterization.
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Laboratory Diagnosis
Sample Collection
Urine is normally a sterile body fluid. However,
unless it is collected properly, it may become
contaminated with flora from the urethra, vagina,
prostate or perineum.
Urine samples are most commonly collected by
obtaining the mid-stream urine flow by the cleancatch technique:
 The priuretheral area and the perineum are
first cleaned
 The first volume of urine is discarded and
washes away the surface commensals from
the distal urethra.
 The midstream specimen is collected for
examination (this specimen is assumed to
represent the true microbiological situation
in the bladder).
Whenever possible, first voided morning urine
should be collected. If not possible, urine should
be allowed to incubate in the bladder for as long
as possible before collection to increase the
number of organisms in the sample.
Urine collection from babies is performed by
adhering a special sterile urine collection bag to
the genitals.
• Midstream urine
collection is
critical in order
to avoid
contamination
with external
flora
• Morning urine
should be
collected if
possible
• Urine specimen
should be
incubated long
enough in the
bladder to
increase the
number of the
organisms/ml
Special collection techniques like suprapubic
aspiration (using a needle for direct aspiration
from the bladder), and catheterization are
reserved to those patients who are unable to
produce a midstream samples.
Transportation of urine specimens to
the laboratory
Urinary tract pathogens grow in urine; therefore,
collected specimens should be processed within
2 hours after collection to achieve accurate
colony counts. In case of delayed transportation
to the laboratory, special urine collection kits
were designed to maintain the bacterial
population in urine at room temperature for 24
hours, however, refrigeration at 4C is widely
accepted as satisfactory for this purpose.
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Culturing microorganisms
Culturing of microorganisms involve two basic
operations:
Isolation: the separation of a particular
microorganism from the mixed population that
exists in nature, and cultivation, the growth of
microbial population in an artificial environment,
culture media under laboratory conditions.
Microorganisms do not require much space for
development, hence an artificial environment can
be created within a test tube, a flask or a petri
dish. The cultural vessel must be rendered
initially sterile, free of any inhibiting substances
and living organisms, and must be protected from
subsequent external contamination.
The primary source of external contamination is
the atmosphere, which always contains floating
microorganisms.
To grow, organisms draw all the substances
which they require for the synthesis of their cell
materials, and the generation of energy from the
environment.
These substances are termed nutrients.
A culture medium must contain all the necessary
nutrients in quantities appropriate to the specific
requirements of the microorganism, for which it is
designed.
However, microorganisms are
extraordinarily
diverse
in
their
specific
physiological properties, and correspondingly in
their specific nutrient requirements.
In constructing a culture medium for any
microorganism, the primary goal is to provide a
balanced mixture of the required nutrients, at
concentrations that will permit good growth. The
selectivity of a culture medium is not determined
solely by the chemical composition of the
medium used but can also be modified by the
variation of other factors such as temperature,
pH,
ionic
strength,
illumination,
etc.
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• Isolation: the
separation of a
particular
microorganism
from a mixed
population
• Cultivation:
the growth of
microbial
population in
an artificial
environment
• Nutrients:
substances
which the
organism
requires
for the synthesis
of their cell
materials
• Culture medium:
a balanced
mixture of the
required
nutrients at
concentrations
that will permit
good growth
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MacConkey Agar (red)
MacConkey #3 (Oxoid) agar is a selective
modification of MacConkey medium suitable for
the detection and enumeration of Gram-negative
bacteria, and also for the detection and isolation
of Salmonella and Shigella species.
• For the detection
and enumeration
of Gram-negative
bacteria
Due to the inclusion of a specially prepared
fraction of bile salts in addition to crystal violet,
this medium gives improved growth differentiation
between coliforms and non-lactose fermenting
organisms, whilst Gram-positive cocci are
completely inhibited by the bile salts.
• Gram-positive
bacteria growth
is inhibited by
the bile salts
Another differential action of MacConkey Agar is
based on fermentation of lactose:
Colonies of organisms capable of fermenting
lactose produce a localized pH drop which,
followed by the absorption of the neutral red, and
imparts a red color to the colony.
A zone of precipitated bile may also be present.
Non-lactose fermenting bacteria do not absorb
the neutral red and the colonies remain colorless
or translucent.
• Lactose
fermenting
bacteria absorb
the neutral red
and appear
pink-red
• Non-lactose
fermenting
bacteria appear
colorless or
translucent
CLED Agar (green)
CLED
(a
dehydrated
Cysteine-LactoseElectrolyte Deficient) agar is designated for the
cultivation and estimation of bacteria from urine.
CLED agar supports the growth of both Gramnegative and Gram-positive bacteria.
• Supports the
growth of all
bacteria in urine
CLED agar is deficient of electrolytes, thus
inhibiting the swarming of proteus speices, which
otherwise would obscure the observation of
colonies.
• Inhibits the
swarming of
proteus
Lactose and indicator bromthymol blue are
included in the medium to detect lactosefermenting bacteria:
Lactic Acid, produced by fermenting bacteria,
causes the indicator bromthymol blue, thus the
agar, to turn yellow. Non-lactose fermenting
bacteria do not cause a color change of the agar
and it remains green to blue.
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• The Agar color
changes to
yellow in the
presence of
fermenting
bacteria
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Diaslide® - Unique
Product
Diaslide® Design
The Diaslide® device is a small transparent plastic
casing with 2 hinged sections facing each other:
One section contains CLED agar, and the other
MacConkey. Diaslide®’s agars are elongated in
order to ensure longitudinal streaking of urine
and isolation of colonies.
Before use the casing is closed and a white
plastic sampler (inoculation) separates the two
parts of the casing.
This specially designed sampler has one handle
on the upper top - to allow the user to pull it for
seeding, and a special tip shaped like a bent fork
on the sampler end.
These latter “fork” tips are dipped into the urine
specimen.
The “fork” tips are designed specially with 2 tips
and 2 bent joints. When pulling the sampler
through the casing devise each agar surface is
streaked by the tip of one of the tips, as well as
by the bent “joint” of the other tip.
• Transparent
plastic casing
• CLED &
MacConkey
facing each
other
• White plastic
sampler for
inoculation
• A very smart
sampler with
2 tips: for
streaking dilution
and 2 joints: for
uniform
spreading
The tip inoculation yields a streaking dilution of
several orders of magnitude, whereas inoculation
by the “joint” yields a relatively uniform spreading
of the sample.
Following inoculation the sampler is discarded
and the case is placed back into its individual
packaging for further incubation.
Up to 20 Diaslide® are put in a special
Polyethylene stand for a vertical incubation.
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Diaslide Packaging
Diaslide
Catalog
No.
CLED/MacConkey 109-01
CNA/MacConkey 110-01
TSA/MacConkey
108-01
COLOREX®
107-01
Packaging
10x10 units
10x10 units
10x10 units
10x10 units
Each Diaslide® is packed in a sterile, flexible,
transparent plastic magazine, tightly reclosed
after sampling, and during incubation.
This flexible casing creates a closed sterile
system for each Diaslide® before use and after
sampling, eliminating potential contamination
from the surroundings, and safer to the user.
The bottom of the flexible case is a small
container, which stores the leaking mucous,
obtained by growing bacteria like Klebsiella.
Collecting the mucous prevents contamination of
the Diaslide® casing and the surroundings in the
incubator, and is more user friendly for the user.
The new flexible casing is easily fitted into a
special foam stand in the incubator reducing
incubator space, and allowing separate
inspection of a single Diaslide®.
Unit package contains 10 Diaslide® units in a
plastic bag.
• A new sterile
flexible
transparent
plastic casing
• Eliminates
potential
contamination
from the
surroundings
• Safer and more
comfortable for
the user
Correct storage
Diaslide®
is stored at Room Temperature
(15-25C) in the original package.
Once a 10-unit package is opened, the remaining
units should be sealed tightly in the bag in order
to maximize shelf life.
Protection from direct light and temperature
fluctuations will ensure the product stability until
the expiration date. Exposure to light may cause
shrinking and fading of the agar media.
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• Store at room
temperature
• Do not expose to
direct light
• Diaslide® MUST
NOT BE
FROZEN.
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Shelf life
Shelf life of Diaslide® is 7 months at room
temperature.
The expiration date is valid if the product is
stored in the plastic bag as indicated in the
instruction manual.
Precautions
Diaslide® should not be used is it exhibits the
following characteristics:
 discoloration
 dehydration
 wrinkling or shrinkage of the agar surface
 microbial growth prior to inoculation
Diaslide® Procedure
Obtaining the Sample
Test reliability is dependent on the method of
specimen collection. Use standard procedure.
The genital area should be cleaned and a first
voided midstream sample should be collected
into a sterile container. The urine should be
inoculated as soon as possible (see ‘sample
collection’ and ‘trasportation’).
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Inoculation Procedure
1. Open the flexible magazine and remove
the Diaslide®, being careful not to touch
the sampler tips.
2. Dip the sampler tips into the urine, making
sure that they are immersed in urine up to
the point where they meet.
3. Hold the device vertically and use your
other hand to pull the sampler through the
casing.
4. Discard the sampler.
5. Put the device back into its flexible
magazine and close it tightly. Write the
patient’s details on a label (or directly on
the plastic) and affix it to the casing.
6. Put the Diaslide® in a vertical position in
the Polyethylene stand supplied.
7. Incubate at 37C for 16-24 hours
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Reading the Results
Following the incubation, examine the Diaslide®
for bacterial growth, which may be evidenced by
visible signs of colony growth on the agar
surface. Every colony results from the natural
multiplication of a single bacterial cell.
1. Colony count
Estimating the bacterial concentration in the urine
sample from the number of colonies grown on the
Diaslide® using the following correlation:
Determining this ratio for E.Coli is as follows:
Concentration
CLED
MacConkey
CFU/ml*
CFU/ml
CFU/ml
103
5
4
4
10
17
15
5
10
110
100
106
~400
~400
*CFU/ml = Colony Forming Unit per 1ml of urine
Compare the colony count to the density chart
provided in the instruction manual
• 1 colony
Results from
1 bacteria
• ‘colony count’ is
the approximate
number of
bacteria per 1ml
of urine.
Notes:
1. When determining the colony count do not
consider size of a given colony but only
the number of colonies.
2. Significant difference in the number of
colonies on one side of the agar vs. the
other may result from an un-even
inoculation. The side with the greater
number of colonies
should be used.
2. Negative Cultures
Culture negative samples can be observed
without opening the Diaslide®, however make
sure to examine the agar against reflecting light.
The lack of reflection indicates confluent growth.
Very small colonies can also be detected in this
manner.
Results of less than 10,000 (104) bacteria per 1ml
are considered to be negative.
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3. Positive Cultures
As a rule, results of >100,000 (105) bacteria per
1ml are considered to be positive. Meaning that
the patient suffers from an infection.
The bacterial growth may consist of very small or
very large colonies. It is important to remember
that only the number of colonies, and not their
size, should be taken into account when
comparing Diaslide® to the colony density chart.
Mixed bacterial growth, which means that
different types of colonies are present, is usually
caused by contamination of the specimen.
is demonstrated in
the
Diaslide®’s
flyer, and in the
instruction for use.
• More than
105 CFU/ml=
Positive
4. Special Consideration
Results between the limits, >104 and <105 are
borderline cases, and should be repeated.
Factors Affecting Colony Count
The following factors may affect bacterial colony
counts in urine specimens:
 Rapid rate of urine flow and frequent
bladder emptying (as occurs in patients with
acute cystitis) - can dilute the bacterial
counts.
 Site of infection is not in the bladder (renal
stones, prostate)
 Urine pH is low (less than 5.0)
 Presence of antibacterial substances in
urine (e.g. high concentration of urea)
 The number of bacteria in one colonyforming unit (e.g. streptococci in chains,
staphylococci in clumps) makes it difficult to
count
 Rate and conditions of growth of different
bacterial species
 Special therapy
5. Colony Morphology
Presumptive identification is based on:
1. typical morphology and color of the colony
2. The agar color.
Typical cultural response on Diaslide® media
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Diaslide® - Positioning &
Strategy
Diaslide® is your superior device
designed to be one step ahead of all
urine culturing devices.
The unique Dip-tip technique differentiates
Diaslide® from all other commercial
dipslides enabling dilution streaking
similar to culture.
Diaslide® combines the sensitivity &
Specificity of the traditional petri dish
culture media, with the convenience of
dipslide, which together with special
unique features makes it the leading
device for UTI testing!
The simple and rapid protocol together
with the see-through view results in a
unique transport packaging position
Diaslide® as a premium high quality
price product, providing easy to use
friendly standardized culture process
from the bedside or doctor’s office up
to
the
clinical
microbiological
laboratory.
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Diaslide® vs. Culture media
Traditional culture employs a streaking dilution of
the urine sample on a solid agar media with a
calibrated loop, providing accurate identification
and quantification of individual colonies.
Nevertheless, one major disadvantage of this
approach is that the urine must be transported to
the microbiology lab, where skilled personnel
then plate it.
Since urine is an excellent medium for bacterial
culture, the number of bacteria present may
increase rapidly if during transportation the
sample is not refrigerated, which, of course,
increases the cost of every test performed.
Total extra-cost for traditional culturing:
1. Expensive skilled personnel for inoculation
2. Transportation to the laboratory
3. Special conditions (refrigeration) for
transported specimens
4. Time consuming preparing the solid agar
in the lab by a technicians.
Diaslide® is a leading product in
comparison to the traditional culture
media with the following advantages:
1. Easy protocol – simple, rapid and handy
2. No skilled personnel are required due to
the simple to use straightforward device
3. Sampling at the site of collection – no
special Lab facilities are required for
preparation
Saves you a lot of space in the incubator due to
its compact design and comfortable incubation
stand
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Diaslide® vs. dipslide
The need for a simple method of applying urine
to solid surface at the collection site has yielded a
variety of approached, in which dipslide is the
most common.
The urine dipslide is a small device with agar
medium usually CLED and MacConkey agars on
each side of an immersible plastic paddle.
Dipslide has previously been shown a cost
effective, simple device, providing comparable
results to those obtained by standard plate
streaking methods.
The
conventional
urine
dipslide
has
disadvantages,
which
comprises
its
usefulness:
1. At concentration of urine, which are higher
than 106 CFU/ml, confluent growth is often
obtained, complicating detection and subculturing.
2. Sampling of urine with Dipslide is
cumbersome and messy: one has to dip
the whole device in urine, dry it on an
absorbance paper for the excess droplets
and put it back into its vial.
3. Dip-Slides are short in comparison to
Diaslide® and therefore isolation of dipslide is not effective.
4. Since large amounts of urine are absorbed
by the immersed agar surfaces, there is
the possibility of false negative results due
to the carry over of inhibitory agents
present in the urine (antibiotics).
5. Sampling of small volumes of urine with
the Dipslide is cumbersome.
6. The condensation, which forms on the
outer vial during incubation, hinders
examination and necessitates unscrewing
of the cap and removing of the attached
slide. Since the majority of the samples
are negative, this comprises the device’s
user friendliness and lengthens the
processing time.
7. Rocket-pneumatic transport of dip-slide
samples to central labs, in big hospitals,
damage the agar causing it to peel off the
paddle.
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Diaslide® is a leading product in
comparison to the traditional culture
with the following advantages:
1. Dilution streaking for isolation and
identification of individual colonies even in
high concentration bacteriuria (107),
especially in geriatric patients.
2. Diaslide® accelerates sub-culturing and
diagnosis by a whole day
3. Diaslide® enables differentiation of mixed
and
contaminated culture
4. Diaslide® secures easier picking
identification and antibiogram.
for
5. Clear and transparent see-through view
enables easier and more precise reading
and interpretation of the results.
6. Low volume specimens can be used; in
fact only 1cm of urine height is necessary
for safe and proper inoculation.
7. Urine sampling directly from babies’ urinebags is possible due to the sampler-fork
design.
8. No inhibition due to the presence of
antibiotics the urine
9. Minimal agar exposure to the environment
during inoculation reduces potential
contamination during the preparation steps
10. Standardized
preparation
technique
enables easy protocol - simple, rapid and
handy
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Who are your Customers?
1. Customers who use the conventional dipslides
today and are aware of the need for
improvements:
Diaslide® is an advanced, new generation
dipslide. The added value features of Diaslide ®
lie in its culture-like capabilities, absent in
conventional dipslides;
 Its ability to isolate single colonies even
in high CFU
 The ability to use small volumes of urine
 Non-inhibition by antibiotics
2. Customers or users who prefer the classical
method to ordinary dipslides based on
performance characteristics:
 Diaslide® in clinically proven. Its
resemblance to culture is well
documented
• Customers who
use the
conventional
dipslides
• Conservative
customers
• Customers with
limited
manpower
3. Customers who require culture quality results
but have limited manpower:
 Diaslide® provides the ability to perform
on site semi-quantitative culture without
the need for qualified personnel
Potential new customers
Diaslide® can be offered to either the doctor or
his assistant at the clinic, the nurse in the hospital
ward or the peripheral laboratory.
• Doctors & Nurse
practitioner’s
office
The receiver of the urine sample directly from the
patient, may perform, without any delay,
Diaslide®’ inoculation, transferring only the
inoculated device to incubation in the laboratory.
Hence, accelerating the process of fresh urine
inoculation, saving time for full diagnosis,
avoiding spillage of urine sample on their way to
the laboratory, avoiding extra cost of refrigerating
samples while being transported to the lab and
benefiting precise sampling with convenient
delivery and high diagnostic performance in the
laboratory.
 Hospital’s
wards
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• Peripheral
laboratory and
satellite
collection sites
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Competition
Summary of competition
All competitive devices are in some way different
forms of no more than the dipslides with similar
design.
The slide is attached to a screw cap and kept in a
plastic container-vial.
One side of the dip-slide is coated with one agar
and the other with another agar.
Common agars for UTI are CLED or EMB and
MacConkey.
 All competitors’
urine devices are
dipslides.
Diaslide® is
unique
The running procedure for the different dipslides
is also similar:
1. The container-vial is opened.and the slide is
pulled out.
2. The slide, exposed to the external
environment, is dipped completely in the
urine sample. The excess urine is allowed
to drain and the last drops are absorbed
with blotting paper.
3. The slide is returned to its container-vial,
tightly capped and incubated for 16-24
hours at 35-37°C.
4. When incubation is done the dip-slide must
be pulled again out of its container-vial,
since condensation on the inner side of
container vial prevent proper inspection and
determination of results.
Diaslide® advantages over the
dipslides are clear:
1. Always kept sterile and not exposed to
external contamination-the agar casing
remains closed.
2. Only the sampler tip is dipped in the urine
sample for real streaking and dilution.
No need to mess with urine and blot excess
urine drops.
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3. No need to open the casing to determine
results since there is no condensation
during incubation.
4. No confluent growth of high concentration
of bacteria.
Competitive Analysis
Feature
Diaslide®
Dipslide
Dilution streaking for
isolation &
identification
—
Clear transparent
and
See-through view
—
Low volume
specimen
—
Less inhibition due
to antibiotics
—
Minimal agar
exposure during
inoculation
Unique transport &
incubation package
Culture
—
—
—
—
Easy & rapid
protocol
—
No skilled personnel
is required
—
Sampling at site of
collection
—
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List of Competitors
URICULT®
Orion Diagnostica,
Finland
The classical and the oldest product on the
market (more than 25 years).
The culture media used on Uricult are:
CLED/MacConkey
CLED/EMB
CLED+Polymyxin/EMB
CLED+Polymyxin/MacConkey
Principle of dipslide as described above.
------------------------------------------------------Uri-Dip®
pHarma-medica of
Scandinavia Ltd. (Canada)
The classical dipslide.
The culture media used on Uri-Dip are:
CLED/MacConkey
CLED/MacConkey w/ocrystal violet
CLED+Polymyxin-B/EMB
CLED+Polymyxin-B/MacConkey
Principle of dipslide as described above.
------------------------------------------------------Uriline® & Uriline ID® bioMérieux,
France
2 products available:
Uriline - a regular dip-slide with
CLED/MacConkey agar
Uriline ID - dip-slide with CLED agar on
one side and ID agar on the
other for identification of E.coli and
Proteus, under fluorescent light,
with 3 enzymes supplied.
Principle of dipslide as described above.
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SOLAR-CULT®
Solar Biological
Ltd. Canada
The classical dip-slide.
The culture media used on SOLAR-CULT is
CLED/MacConkey
Principle of dipslide as described above.
------------------------------------------------------Contact-Slide®
Roche, Switzerland
A special triangles slide.
The culture media used on Contact-Slide is
CLED/MacConkey/?
Principle of dipslide as described above.
------------------------------------------------------D.G.U. ®
Sanofi-Pasteur,
France
The classical dipslide.
The culture media used on D.G.U. is
CLED/MacConkey
Principle of dipslide as described above.
------------------------------------------------------LA-SLIDE Labobasi SA, Italy
A special dip-slide with 3 culture media on a star
shaped paddle.
The culture media used are:
CLED/MacConkey/Cetrimide
Principle of dipslide as described above.
-------------------------------------------------------
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Micro-Dip 5
Diatec tecnologie
Diagnostiche, Italy
A special dip-slide with 5 culture media on a star
shaped paddle.
The culture media used are:
CLED/MacConkey/Cetrimide/Slanetz Bartley/
Principle of dipslide as described above.
------------------------------------------------------Pentadip®
Promadi Diagnostics,
Italy
A special dip-slide with 5 culture media on a star
shaped paddle.
Principle of dipslide as described above.
------------------------------------------------------DipStreak®
Novamed Ltd.,
Israel
The culture media used on DipStreak are:
CLED/MacConkey
TSA-Blood/MacConkey
Columbia+CNA/MacConkey
UriSelect3/MacConkey
A ring with elongated prongs is attached to the
paddle, only the ends of the prongs are dipped
into the urine sample. The prongs inoculate the
agar surface resulting in a series of streaks
enabling bacterial quantifying.
However, the device is shorter than Diaslide®
so isolation of colonies is limited.
-------------------------------------------------------
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Robobact®
DIESSE, Italy
An automatic system for isolation of urine
pathogens without opening of the sample
container.
2 devices + automated system:
URISET - special collection and transport
device
URIBACT - special development and
isolation device
A special tip designed for streaking the media
automaticaly without exposing the culture to the
external environment.
A very complicated device.
------------------------------------------------------Dip-Slide®
Oxoid, England
A classical dip-slide with one advantage: a
flexible hinge with internal handle allows the
entire media surface to be pressed evenly onto
the test surface.
Principle of dipslide as described above.
------------------------------------------------------Dip ‘N Count® Starplex scientific
Canada
The classical dip-slide.
The culture media used on Dip ‘N Count® are:
CLED/MacConkey
CLED/EMB
CLED+Polymyxin-B Sulphate/MacConkey
Principle of dipslide as described above.
-------------------------------------------------------
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Troubleshooting
1. Question:
Bacteria did not grow on Diaslide®, what is the
reason for this?
Answer:
1. No bacteria in the urine sample: the urine is
sterile or the bacteria count is under the limit
of detection.
2. The sampler was damaged, streaking tracks
of the sampler on the agars are absent, and
therefore the inoculation of bacteria was not
effective.
3. Diaslide® was not stored properly and the
agar was dehydrated and shriveled,
therefore the sampler’s tips and joints did
not touch the agar during inoculation.
2. Question:
The color of the agar is faded, what is the reason
for this?
Answer:
1. Direct exposure to sunlight. If the Diaslide®
is still within the expiry date you can still use
the device.
2. Diaslide® was not stored properly temperature too high (above 25°C).
3. Question:
The agar is dried and shriveled, what is the
reason for this?
Answer:
1. Diaslide® was not stored properly.
2. Package was not reclosed after using a few
devices.
3. The device has passed the expiry date. Do
not use dried and shriveled devices.
4. Question:
Diaslide® was contaminated before use.
Answer:
In rare cases contamination of fungi and bacterial
may have occurred during production.
Do not use the contaminated device, return to
manufacturer.
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5. Question:
Colonies grew on CLED agar only, is there a
problem of the device?
Answer:
1. Gram-positive bacteria do not grow on
Macconkey agar #3 (Oxoid).
2. MacConkey agar dehydrates faster than the
CLED agar. Therefore its height may drop
so the sampler does not streak it properly.
Look for the streaking tracks of the sampler
on the MacConkey agar. Note: this problem
is rare and might occur if the devices are
not properly stored.
Use an intact device.
6. Question:
I think that I did not dip the sampler properly.
Could I re-dip the sampler in the urine sample
again?
Answer:
You can dip the sampler twice only if you still did
not pull it through the agar.
Never pull the sampler twice.
Re-dipping and re-pulling the sampler may tear
the agar when you push it back.
7. Question:
Sampling at emergency rooms (ER,) or at
satellite collection site, was done during the night
while the microbiology laboratory was shut, and
therefore incubation in 37°C was impossible.
Could we use this device for records?
Answer:
Yes.
ER nurses (or peripheral laboratory technicians)
should be instructed to incubate the Diaslide ®
vertically at room temperature until lab facilities
are available.
However, exact time of sampling should be
labeled on the device, so the laboratory will
continue with an additional shorter time of
incubation to ensure adequate incubation time.
8. Question:
The device was received with the sampler fork
not properly in position, could we slide it into
place and use this device?
Answer:
No.
Re-sliding the sampler into place may tear the
agar when you push it back.
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Published Articles
1. Rosenberg M., S.A Berger, M.Barki, S.Goldberg, A.Fink, and A.Miskin,
1992. Initial Testing of a Novel Urine Culture Device. Journal of
Clinical Microbiology, 30(10) 2686-2691.
2. Ferguson J., J.Tanner and M.Miller, 1995. Evaluating of a New,
Semiquantitative Screening Culture Device for Urine Specimens.
Journal of Clinical Microbiology,30(5), 1351-1353
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Package Inserts
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Novamed Ltd.
DIASLIDE Urine Culture Device
C.L.E.D. / MacConkey Agar
Catalog No. DS-101
INTENDED USE
Diaslide Urine Culture Device (UCD) is a semi-quantitative screening culture device for detecting,
enumerating and identifying specific bacteria in urine1. The device is intended for use in physician’s office
laboratories (POL) and clinical laboratories as an aid in the diagnosis of urinary tract infection (UTI).
SUMMARY AND EXPLANATION
Diaslide UCD consists of a transparent, hinged plastic casing containing face-to-face plates of CLED and
MacConkey agars with a plastic sampler having two bent tips located between.
CLED agar supports growth of both Gram-positive and Gram-negative bacteria, providing color changes
that help in preliminary identification, especially of lactose-fermenting organisms, which change the color of
the medium from green to yellow2. The most significant lactose-fermenting bacteria involved in urinary tract
infections
are
Escherichia
coli,
Klebsiella,
Enterobacter
and
Enterococcus.
CLED agar also supports Proteus and Pseudomonas, which do not ferment lactose. However, the low salt
concentration of CLED agar inhibits the swarming of Proteus, which would otherwise obscure the
observation of colonies.
MacConkey agar is a selective medium, giving an excellent differentiation between coliforms and nonlactose fermenters with inhibition of Gram-positive micrococci3. Most urinary tract pathogens grow on it,
whereas most contaminants are inhibited.
Current routine methods for bacteriological examination of urine are the classic Petri dish culture method4
and the and the dipslide technique5. Diaslide UCD combines the advantages of both techniques, enabling
bacterial enumeration and isolation following a simple, user-friendly procedure.
PRINCIPLES OF THE PROCEDURE
The two bent tips of the sampler are dipped into the urine specimen and take up a standard volume of
urine. The sampler is then pulled out through the casing, simultaneously inoculating the surfaces of both
media with a streaking dilution. As a consequence, the number of bacteria deposited on the media is in
direct proportion to the number of bacteria present in the specimen6. Following incubation, the number of
bacterial colonies cultures is compared with the Colony Density Chart to determine the level of bacteria in
the urine sample.
KIT CONTENTS
Diaslide UCD contains CLED and MacConkey agars.
CLED Medium
MacConkey Agar
Formula per liter
Formula per liter
Lab-Lenco Powder
3g
Peptone
Peptone
Tryptone
L-Cystine
Lactose
Agar
BromoThymol Blue
Final pH 7.3  0.2 at 25C
4g
4g
0.128 g
10 g
15 g
0.02 g
Lactose
Bile salts No. 3
Sodium Chloride
Agar
Neutral Red
Crystal Violet
Final pH 7.2  0.2 at 25C
20 g
10 g
1.5 g
5g
15 g
0.03 g
0.001 g
MATERIAL REQUIRED BUT NOT PROVIDED
Incubator (37  1C)
Incubation Stand
WARNING AND PRECAUTIONS
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1. For In Vitro Diagnostic Use.
2. Use aseptic technique and established laboratory procedure in handling and disposing of infectious
material.
STORAGE
1. Store Diaslide UCD at 6-25C.
2. Protect contents from direct light to ensure product stability through the expiration date.
3. Upon opening the bag, store unused Diaslide UCD units by rolling the open end of the bag and
sealing it tightly with tape over the entire length of the opening.
EXPIRATION DATE
1. The expiration date applies to the product in its intact container when stored as directed.
2. Do not use Diaslide UCD exhibiting any of the following characteristics: discoloration, dehydration,
wrinkling or shrinkage of an agar surface; microbial growth prior to inoculation; or an atypical cultural
response in Quality Control procedures.
SPECIMEN COLLECTION
Cleanse the genital area and collect a midstream urine specimen in a clean container. Inoculate the urine
as soon as possible following collection.
If storage of the urine specimen is necessary, maintain the
specimen at 4C in a closed sterile container. Storage time should not exceed two hours.
PROCEDURE
1. Remove Diaslide UCD from its packaging, being careful not to touch the sampler tips (Fig. 1).
2. Dip the sampler tips into the thoroughly mixed urine sample, making sure that both tips are immersed up
to the point where they meet (Fig. 2).
3. Holding Diaslide UCD vertically with one hand, use the other hand to draw the sampler up through the
casing in a straight manner (Fig. 3). Discard the sampler.
4. Write patient ID directly on the Diaslide casing, or apply an in-house ID label, as required.
5. Return the Diaslide casing into its packaging, close firmly and stand upright in the incubation stand
(Fig. 4).
6. Incubate in a vertical position at 37C for 18-24 hours in the incubation stand.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
RESULTS
Following incubation, examine Diaslide UCD for bacterial growth, which may be evidenced by visible
colonies on the agar surface. Since each colony results from growth of a single bacterial cell, and since the
sampler takes up a standard volume of urine, the numbers of colonies can indicate the “colony count” of the
specimen,
the
approximate
number
of
bacteria
per
ml
(CFU/ml)
of
urine.
Culture-negative samples can be observed without opening the Diaslide casing. If microbial growth is
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present, open the Diaslide UCD casing and match the “colony density” on the agar surface with the
printed illustration it most closely resembles on the Colony Density Chart (See below).
TABLE 1: The relationship between bacterial concentration (CFU/ml) and the approximate colony
count of the Colony Density Chart.
ORGANISM
CFU/ml
CLED Agar
MacConkey Agar
103
5
4
Escherichia coli
4
10
17
15
105
110
100
106
-400
-400
INTERPRETATION OF RESULTS
Bacterial count
As a rule, tests yielding 105 CFU/ml are regarded as positive (significant level of organism), 104 CFU/ml
as negative, and between 104 and 105 CFU/ml as borderline, which calls for a repeat assessment7.
Symptomatic patients having colony counts less than 10 5 CFU/ml require evaluation based on clinical
information9, 10. Many factors, such as use of antimicrobial therapy, time of urine incubation in the bladder
(e.g., first voided urine), and proper specimen collection, may influence the colony count obtained. In all
cases, the physician must be the final judge of the proper interpretation of Diaslide UCD test results.
COLONY DENSITY CHART
Colony Morphology
Presumptive identification is based on typical morphology and colony color, particularly on CLED agar.
TABLE 2. Typical colonial morphology on Diaslide UCD media.
ORGANISM
CLED Agar
MacConkey Agar
Yellow, darker center
Pink-red
Escherichia coli
Mucous yellow
Pink-red
Klebsiella pneumoniae
Mat green-blue
Clear
Pseudomonas aeruginosa
Translucent, gray-blue
Translucent
Proteus mirabilis
Clear green-blue
Clear
Proteus Vulgaris
Dark Yellow
No growth
Staphylococcus aureus
White or pale yellow
No growth
Staphylococcus epidermidis
Yellow
No growth
Streptococcus faecalis
Since other organisms also grow on these media, full biochemical analysis and antimicrobial susceptibility
testing are required for complete identification.
USER QUALITY CONTROL
Quality control tests are performed on each lot of Diaslide at the time of manufacture. Product users who
wish to perform their own quality control may use the following procedure.
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1. Prepare a suspension (104-105 CFU/ml) of each of the following organisms in culture-negative urine
from a healthy individual. Confirm the exact organism concentration by inoculating 10l with a
calibrated loop on reference plates of CLED and MacConkey agar.
2. Test according to the PROCEDURE.
TYPICAL CULTURAL RESPONSE ( after 24 hours at 37C)
ORGANISM
CLED Agar
MacConkey Agar
ATCC
25922
Yellow colonies, medium becoming yellow Pink colonies
Escherichia coli
Yellow colonies, yellow medium
Inhibited
Staphylococcus aureus 25923
Translucent colonies, medium becoming
Translucent,
Proteus mirabilis
12453
blue
colorless colonies
3. If the device does not support the expected growth of organisms, it has deteriorated and should not be
used.
LIMITATIONS OF THE PROCEDURE
1. Diaslide UCD is a presumptive screening test. If the physician concludes that it clinically indicates, full
biochemical identification of causative organism(s) and antimicrobial susceptibility testing, should be
performed.
2. Diaslide UCD is capable of detecting bacteriuria concentrations as low as 1000 CFU/ml of urine. The
Colony Density Chart allows the reporting of colony counts to the nearest power of 10.
When used as directed, an overall correlation of 95% is obtained when Diaslide UCD colony count results
are compared to conventional pour plate methods.
3. If bacterial growth is mixed, i.e., made up of different kinds of colonies, repeat the test since this is most
likely due to contamination. If the same results are repeated, testing of a second urine sample is
recommended.
4. Infants and certain patients may have a true infection even if the bacterial count is less than 10 5 CFU/ml.
Final interpretation of such results should be evaluated based on clinical information. Protocols for
individual laboratories must be established based on close cooperation between the medical and laboratory
staff. The guidelines are based on the principle that four factors (number of isolates, density of isolates,
type of specimen, and clinical information) must be considered to asses the significance of an isolate.
Clinical information may alter the physician’s final interpretation of culture results 9.
5. If bacterial content in a urine specimen is above 107 CFU/ml, no single colony can be isolated because
of confluent growth, even in the “isolation track”. A standard quantitative urine culture should be performed.
PERFORMANCE CHARACTERISTICS
Diaslide UCD test was evaluated relative to the standard plate culture method, which is the currently
accepted method for detecting organisms in urine. Specimens were mixed by inversion and 10l of urine
was delivered with a calibrated loop to the surface of MacConkey agar with 5% sheep blood. Plates were
incubated under 5% CO2 at 35C for 18-24 hours, at which time the number of colonies were counted.
Diaslide UCD test results were also compared with those of another commercially available urine
dipslide. Urine specimens were obtained from patients from a 700-bed hospital, 30% from geriatric and
chronic patients and the remainder from other wards and outpatient clinics. Specimens were randomly
selected and screened by a rapid UTI screen,8 specimens testing positive (n=473) were employed in this
study.
TABLE 3: The sensitivity and specificity of Diaslide vs. the Petri dish method at a cut-off 105 CFU/ml
Diaslide UCD
Borderline
Positive
Negative
104 - 105
Total
105 CFU/ml
104 CFU/ml
CFU/ml
Positive
206
4
2
212
CULTURE
Negative
5
226
5
236
Borderline
3
5
17
25
Total
214
235
24
473
Relative Sensitivity = 98.1% Relative Specificity = 97.8%
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Thirty two (32) of 473 samples (6.8%) were found borderline by at least one of the methods. Seventeen
(17) of those 32 samples (53%) were found borderline by both methods.
TABLE 4: The sensitivity and specificity of Diaslide UCD vs. the Petri dish method at a cut-off of 104
CFU/ml
Diaslide UCD
Borderline
Positive
Negative
103 - 104
Total
104 CFU/ml
103 CFU/ml
CFU/ml
Positive
238
4
1
243
CULTURE
Negative
4
157
3
164
Borderline
12
43
11
66
Total
254
204
15
473
Relative Sensitivity = 98.3% Relative Specificity = 97.5%
Seventy (70) of 473 samples (14.8%) were found borderline by at least one of the methods. Eleven (11) of
those 70 samples (15.7%) were found borderline by both methods.
TABLE 5: The sensitivity and specificity of Diaslide UCD vs. the Uricult at a cut-off 105 CFU/ml
Diaslide UCD
Borderline
Positive
Negative
104 - 105
Total
105 CFU/ml
104 CFU/ml
CFU/ml
Positive
213
6
3
222
URICULT
Negative
1
229
7
237
Borderline
0
0
14
14
Total
214
235
24
473
Relative Sensitivity = 97.3% Relative Specificity = 99.6%
Twenty four (24) of 473 samples (5%) were found borderline by at least one of the methods. Fourteen (14)
of those 24 samples (58.3%) were found borderline by both methods.
TABLE 6: The sensitivity and specificity of Diaslide UCD vs. the Uricult at a cut-off of 104 CFU/ml
Diaslide UCD
Borderline
Positive
Negative
103 - 104
Total
104 CFU/ml
103 CFU/ml
CFU/ml
Positive
250
5
3
258
URICULT
Negative
2
191
1
194
Borderline
Total
2
254
8
204
11
15
21
473
Relative Sensitivity = 98% Relative Specificity = 99%
Twenty five (25) of 473 samples (5.3%) were found borderline by at least one of the methods. Eleven (11)
of those 25 samples (44%) were found borderline by both methods.
Reproducibility among Personnel in Physician Office Laboratory
Reproducibility among personnel was determined by running the same spiked urine samples at three
different physician’s office locations (A,B,C) at the same time. Each sample was run in duplicate.
Suspensions of three different bacteria, at four different concentrations each were prepared; actual
concentration was confirmed by the standard culture method.
These suspensions were used by three different technicians for determining bacterial concentration using
Diaslide UCD as instructed in the test procedure. The approximate count was read from the Colony
Density Chart. If only a few colonies grew, the exact number of colonies was counted, and the approximate
concentration was determined from TABLE 7.
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TABLE 7. Comparison of test results obtained at three different test locations.
INTERPRETATION OF RESULTS ACCORDING TO THE
COLONY DENSITY CHART
Technician A
Technician B
Technician C
1.2 x 103
103 Neg
<103 Neg
103 Neg
103 Neg
<103 Neg
103 Neg
4
4
4
1.2 x 10
10 Neg
10 Neg
103 Neg
4
4
Escherichia
10 Neg
10 Neg
103 Neg
5
5
5
coli
1.2 x 10
10 Pos
10 Pos
105 Pos
105 Pos
105 Pos
105 Pos
6
6
6
1.2 x 10
10 Pos
10 Pos
106 Pos
6
6
10 Pos
10 Pos
106 Pos
2
3
3
3 x 10
10 Neg
<10 Neg
103 Neg
103 Neg
103 Neg
103 Neg
3
3
3
3 x 10
10 Neg
<10 Neg
103 Neg
3
3
Staphylococcus
10 Neg
10 Neg
103 Neg
4
4
3
aureus
3 x 10
10 Neg
10 Neg
103 Neg
104 Neg
<103 Neg
103 Neg
5
5
5
3 x 10
10 Pos
10 Pos
105 Pos
5
5
10 Pos
10 Pos
105 Pos
1.1 x 103
104 Neg
103 Neg
103 Neg
3
3
10 Neg
10 Neg
103 Neg
4
5
4
1.1 x 10
10 Pos
10 Neg
104 Neg
5
4
Proteus
10 Pos
10 Neg
104 Neg
5
5
6
5
mirabilis
1.1 x 10
10 -10 Pos
10 Pos
105 Pos
105-106 Pos
105 Pos
105 Pos
6
6
6
1.1 x 10
10 Pos
10 Pos
106 Pos
6
6
10 Pos
10 Pos
106 Pos
* As determined by the standard culture method.
ORGANISM
CFU/ml*
Conclusion
Reproducibilty among personnel was calculated. In only one of 12 samples (each in duplicate) there was a
disagreement between technicians’ results. Seventy (70) of 72 determinations exhibited complete
agreement between technicians’ results (70/72 x 100 = 97.2% agreement). The results correlated well with
the concentration of bacteria determined by culture.
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BIBLIOGRAPHY
1. Rosenberg, M., S.A. Berger, M. Barki, S. Goldberg, A. Fink and A. Miskin. 1992. Initial Testing of a
Novel urine Culture Device. Journal of Clinical Microbiology. p. 2686-2691.
2. The Oxoid Manual, 7th Edition. 1995. Oxoid Unipath, Basingstoke, England. 2-75: 2-76
3. The Oxoid Manual, 7th Edition. 1995. Oxoid Unipath, Basingstoke, England. 2-144:2-145.
4. Degre, M. 1970. Quantitative and semiquantitative methods for detection of bacteriuria. Translated from
Tidsskrift for Den norske laegeforening 90: 27-30.
5. McAllister, T.A. 1973. The day of the Dipslide. Nephron 11:123-133.
6. Mackey, J.P. and G.H. Sandys. 1965. Laboratory diagnosis of infections of the urinary tract in general
practice by means of a dip-inoculum transport medium. British Medical Journal. 2:1286-1288.
7. Kass, E.H. 1957. Bacteriuria and the diagnosis of infections of the urinary tract. Arch. Int. Med. 100:709714.
8. Berger, S.A., B. Bogokowsky, and C. Block. 1990. Rapid screening of urine for bacteria and cells by
using a catalase reagent. J. Clin. Micro. 28:1066-1067.
9. Clarridge, J.E., M.T. Pezzlo, and K.L. Vosti. 1987. Laboratory diagnosis of urinary tract infections. In
Cumitech 2A. American Society for Microbiology, Washington D.C.
10. Isenberg, H.D. and R.F. D’Amaato. 1985. Indigenous and pathogenic microorganisms of humans, p. 3031. In E.H. Lennette, A. Ballows, W.J. Hausler, Jr., and H.J. Shadomy, Manual of Clinical Microbiology,
Washington, D.C.
PACKAGING
Diaslide CLED / MacConkey Urine Culture Device 10 tests
10 x 10 tests DS-101
Manufactured by:
Novamed Ltd.
28, Pierre Koenig Street
Talpiot Industrial Area
POB 53231 Jerusalem 91531
ISRAEL
Tel.: +972.2 6781861
Fax: +972.2 6781852
E-mail: [email protected]
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Novamed Ltd.
DIASLIDE Urine Culture Device
Columbia CNA / MacConkey Agar
Catalog No. DS-102
INTENDED USE
Diaslide Urine Culture Device (UCD) is a semi-quantitative screening culture device for detecting,
enumerating and identifying specific bacteria in urine. The device is intended for use in physician’s office
laboratories (POL) and clinical laboratories as an aid in the diagnosis of urinary tract infection (UTI).
SUMMARY AND EXPLANATION
Diaslide UCD (Columbia CNA/MacConkey) consists of a transparent, hinged plastic casing containing
face-to-face agar plates. One plate contains Columbia CNA agar while the other contains MacConkey agar
with a plastic sampler having two bent tips located between the two agar plates.
MACCONKEY AGAR is a selective and differential medium for detection of coliform organisms and enteric
pathogens. The concentration of bile salts in this medium is relatively low in comparison with other enteric
plating medium; therefore selectivity for gram-negative bacteria is not as great as in some other
formulations.
Crystal violet inhibits gram-positive microorganisms, especially enterococci and
staphylococci. Differentiation of enteric microorganisms is achieved by the combination of lactose and the
neutral red indicator. Colorless or pink to red colonies are produced depending upon the ability of the
isolate to ferment lactose.
COLUMBIA CNA AGAR supplemented with 5% sheep blood is a selective and differential medium used for
the isolation and differentiation of gram-positive microorganisms from clinical and non-clinical materials.
Current routine methods for bacteriological examination of urine are the classic Petri dish culture method
and the dipslide technique. Diaslide UCD combines the advantages of both techniques, enabling bacterial
enumeration and isolation following a simple, user-friendly procedure.
PRINCIPLES OF THE PROCEDURE
The two bent tips of the sampler are dipped into the urine specimen and take up a standard volume of
urine. The sampler is then pulled out through the casing, simultaneously inoculating the surfaces of both
media with a streaking dilution. As a consequence, the number of bacteria deposited on the media is in
direct proportion to the number of bacteria present in the specimen. Following incubation, the number of
bacterial colonies cultures is compared with the Colony Density Chart to determine the level of bacteria in
the urine sample.
KIT CONTENTS
Diaslide UCD REF DS-102 units contain Columbia CNA agar and MacConkey agar.
MATERIALS REQUIRED BUT NOT PROVIDED
Incubator (37  1C)
Incubation Stand
WARNING AND PRECAUTIONS
1. For In Vitro Diagnostic Use.
2. Use aseptic technique and established laboratory procedure in handling and disposing of infectious
material.
STORAGE
1. Store Diaslide UCD at 2-8C: refer to package label.
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2. Protect contents from direct light to ensure product stability through the expiration date.
3. Upon opening the bag, store unused Diaslide UCD units by rolling the open end of the bag and
sealing it tightly with tape over the entire length of the opening.
EXPIRATION DATE
1. The expiration date applies to the product in its intact container when stored as directed.
2. Do not use Diaslide UCD exhibiting any of the following characteristics: discoloration, dehydration,
wrinkling or shrinkage of an agar surface; microbial growth prior to inoculation; or an atypical cultural
response in Quality Control procedures.
SPECIMEN COLLECTION
Cleanse the genital area and collect a midstream urine specimen in a clean container. Inoculate the urine
as soon as possible following collection.
If storage of the urine specimen is necessary, maintain the specimen at 4 C in a closed sterile container.
Storage time should not exceed two hours.
PROCEDURE
Materials Provided
Diaslide Urine Culture Device
1. Remove Diaslide UCD from its packaging, being careful not to touch the sampler tips (Fig. 1).
2. Dip the sampler tips into the thoroughly mixed urine sample, making sure that both tips are immersed
up to
the point where they meet (Fig. 2).
3. Holding Diaslide UCD vertically with one hand, use the other hand to draw the sampler up through the
casing in a straight manner (Fig. 3). Discard the sampler.
4. Write patient ID directly on the Diaslide casing, or apply an in-house ID label, as required.
5. Return the Diaslide casing into its packaging, close firmly and stand upright in the incubation stand
(Fig. 4).
6. Incubate in a vertical position at 37C for 18-24 hours in the incubation stand.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
RESULTS
Following incubation, examine Diaslide UCD for bacterial growth, which may be evidenced by visible
colonies on the agar surface.
Since each colony results from growth of a single bacterial cell, and since the sampler takes up a standard
volume of urine, the numbers of colonies can indicate the “colony count” of the specimen, the approximate
number of bacteria per ml (CFU/ml) of urine. Culture-negative samples can be observed without opening
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the Diaslide casing. If microbial growth is present , open the Diaslide UCD casing and match the “colony
density” on the agar surface with the printed illustration it most closely resembles on the Colony Density
Chart (See below).
TABLE 1: The relationship between bacterial concentration (CFU/ml) and the approximate colony count of
the Colony Density Chart.
ORGANISM
E. coli
CFU/ml
103
104
105
106
MacConkey Agar
4
15
100
-400
INTERPRETATION OF RESULTS
In the past, the presence of > 105 CFU/ml was regarded as a positive result while < 104 CFU/ml was
negative. From 104 to 105 CFU/ml were considered borderline cases, which called for repeat examination.
Today, the trend is for specific medical departments such as Urology, Nephrology and Pediatrics to report
down to 103 CFU/ml. Use the attached colony density chart for reporting results. If bacterial growth yields
three or more different kinds of colonies, this is most likely due to contamination. The test should be
repeated.
Preliminary identification of the bacteria can be made based on type and color of colonies. For example:
Organism
E. coli
K. pneumoniae
P. aeruginosa
P. vulgaris
S. aureus
S. epidermidis
E. faecalis
MacConkey
Pink red
Pink red
Colorless
Colorless
No growth
No growth
No growth
Columbia CNA
No growth
No growth
No growth
Partial to total inhibition
White to gold
White
Opaque, grayish
Observe the bacterial growth on both agars. If bacterial growth is present on either Columbia CNA or
MacConkey, but not on both, count the colonies and compare to the chart or to the guidelines for
enumeration of bacteria. If bacterial growth is present on both media, relate to the medium yielding the
highest count. If both media yield approximately the same count, double the total count, after ruling out that
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the same organism is growing on both media, e.g., certain pseudomonades are only partially inhibited on
Columbia CNA.
USER QUALITY CONTROL
Quality control tests are performed on each lot of Diaslide at the time of manufacture. Product users who
wish to perform their own quality control may use the following procedure.
1. Prepare a suspension (104-105 CFU/ml) of each of the following organisms in culture-negative
urine from a healthy individual. Confirm the exact organism concentration by inoculating 10 l with
a calibrated loop on reference plates of Columbia CNA and MacConkey agars.
2. Test according to the PROCEDURE.
3. If the device does not support the expected growth of organisms, it has deteriorated and should
not be used.
TYPICAL CULTURAL RESPONSE (after 24 hours at 37C)
ORGANISM
P. aeruginosa
S. aureus
S. pneumoniae
S. pyogenes
ATCC
27853
25923
6305
19615
Columbia CNA Agar
Inhibited
Beta hemolysis
Alpha hemolysis
Beta hemolysis
ORGANISM
E. coli
S. aureus
P. mirabilis
ATCC
25922
25923
12453
MacConkey Agar
Pink colonies
Inhibited
Translucent, colorless
colonies
LIMITATIONS OF THE PROCEDURE
1. Diaslide UCD is a presumptive screening test. If the physician concludes that it clinically indicates, full
biochemical identification of causative organism(s) and antimicrobial susceptibility testing, should be
performed.
2. Diaslide UCD is capable of detecting bacteriuria concentrations as low as 1000 CFU/ml of urine. The
Colony Density Chart allows the reporting of colony counts to the nearest power of 10.
When used as directed, an overall correlation of 95% is obtained when Diaslide UCD colony count results
are compared to conventional pour plate methods.
3. If bacterial growth is mixed, i.e., made up of different kinds of colonies, repeat the test since this is most
likely due to contamination. If the same results are repeated, testing of a second urine sample is
recommended.
4. Infants and certain patients may have a true infection even if the bacterial count is less than 105
CFU/ml. Final interpretation of such results should be evaluated based on clinical information.
Protocols for individual laboratories must be established based on close cooperation between the
medical and laboratory staff. The guidelines are based on the principle that four factors (number of
isolates, density of isolates, type of specimen, and clinical information) must be considered to asses
the significance of an isolate. Clinical information may alter the physician’s final interpretation of culture
results9.
5. If bacterial content in a urine specimen is above 107 CFU/ml, no single colony can be isolated because
of confluent growth, even in the “isolation track”. A standard quantitative urine culture should be
performed.
PERFORMANCE CHARACTERISTICS
Diaslide UCD test was evaluated relative to the standard plate culture method, which is the currently
accepted method for detecting organisms in urine. Specimens were mixed by inversion and 10l of urine
was delivered with a calibrated loop to the surface of MacConkey agar with 5% sheep blood. Plates were
incubated under 5% CO2 at 35C for 18-24 hours, at which time the number of colonies were counted.
Diaslide UCD test results were also compared with those of another commercially available urine dipslide.
Urine specimens were obtained from patients from a 700-bed hospital, 30% from geriatric and chronic
patients and the remainder from other wards and outpatient clinics. Specimens were randomly selected
and screened by a rapid UTI screen, specimens testing positive (n=473) were employed in this study.
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TABLE 3: The sensitivity and specificity of Diaslide UCD vs. the Petri dish method at a
CFU/ml.
Diaslide UCD
Positive
Negative
Borderline
104 - 105 CFU/ml
105 CFU/ml
104 CFU/ml
Positive
206
4
2
CULTURE
Negative
5
226
5
Borderline
3
5
17
Total
214
235
24
Relative Sensitivity = 98.1% Relative Specificity = 97.8%
Thirty-two (32) of 473 samples (6.8%) were found borderline by at least one of the methods.
(17) of those 32 samples (53%) were found borderline by both methods.
cut-off 105
Total
212
236
25
473
Seventeen
TABLE 4: The sensitivity and specificity of Diaslide UCD vs. the Petri dish method at a cut-off of 104
CFU/ml.
Diaslide UCD
Positive
Negative
Borderline
Total
103 - 104 CFU/ml
104 CFU/ml
103 CFU/ml
Positive
238
4
1
243
CULTURE
Negative
4
157
3
164
Borderline
12
43
11
66
Total
254
204
15
473
Relative Sensitivity = 98.3% Relative Specificity = 97.5%
Seventy (70) of 473 samples (14.8%) were found borderline by at least one of the methods. Eleven (11) of
those 70 samples (15.7%) were found borderline by both methods.
TABLE 5: The sensitivity and specificity of Diaslide UCD vs. the Uricult at a cut-off 105 CFU/ml.
Diaslide UCD
Positive
Negative
Borderline
Total
104 - 105 CFU/ml
105 CFU/ml
104 CFU/ml
Positive
213
6
3
222
URICULT
Negative
1
229
7
237
Borderline
0
0
14
14
Total
214
235
24
473
Relative Sensitivity = 97.3% Relative Specificity = 99.6%
Twenty four (24) of 473 samples (5%) were found borderline by at least one of the methods. Fourteen (14)
of those 24 samples (58.3%) were found borderline by both methods.
TABLE 6: The sensitivity and specificity of Diaslide UCD vs. the Uricult at a cut-off of 104 CFU/ml.
Diaslide UCD
Positive
Negative
Borderline
Total
103 - 104 CFU/ml
104 CFU/ml
103 CFU/ml
Positive
250
5
3
258
URICULT
Negative
2
191
1
194
Borderline
2
8
11
21
Total
254
204
15
473
Relative Sensitivity = 98% Relative Specificity = 99%
Twenty five (25) of 473 samples (5.3%) were found borderline by at least one of the methods. Eleven (11)
of those 25 samples (44%) were found borderline by both methods.
Reproducibility among Personnel in Physician Office Laboratory
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Reproducibility among personnel was determined by running the same spiked urine samples at three
different physician’s office locations (A,B,C) at the same time. Each sample was run in duplicate.
Suspensions of three different bacteria, at four different concentrations each were prepared; actual
concentration was confirmed by the standard culture method.
These suspensions were used by three different technicians for determining bacterial concentration using
Diaslide UCD as instructed in the test procedure. The approximate count was read from the Colony
Density Chart. If only a few colonies grew, the exact number of colonies was counted, and the approximate
concentration was determined from TABLE 1.
TABLE 7. Comparison of test results obtained at three different test locations.
INTERPRETATION OF RESULTS ACCORDING TO THE
ORGANISM
CFU/ml*
COLONY DENSITY CHART
Technician A
Technician B
Technician C
E. coli
1.2 x 103
103 Neg
<103 Neg
103 Neg
103 Neg
<103 Neg
103 Neg
4
4
4
1.2 x 10
10 Neg
10 Neg
103 Neg
4
4
10 Neg
10 Neg
103 Neg
5
5
5
1.2 x 10
10 Pos
10 Pos
105 Pos
105 Pos
105 Pos
105 Pos
6
6
6
1.2 x 10
10 Pos
10 Pos
106 Pos
6
6
10 Pos
10 Pos
106 Pos
2
3
3
S. aureus
3 x 10
10 Neg
<10 Neg
103 Neg
103 Neg
103 Neg
103 Neg
3
3
3
3 x 10
10 Neg
<10 Neg
103 Neg
3
3
10 Neg
10 Neg
103 Neg
4
4
3
3 x 10
10 Neg
10 Neg
103 Neg
104 Neg
<103 Neg
103 Neg
5
5
5
3 x 10
10 Pos
10 Pos
105 Pos
5
5
10 Pos
10 Pos
105 Pos
P. mirabilis
1.1 x 103
104 Neg
103 Neg
103 Neg
3
3
10 Neg
10 Neg
103 Neg
4
5
4
1.1 x 10
10 Pos
10 Neg
104 Neg
5
4
10 Pos
10 Neg
104 Neg
1.1 x 105
105-106 Pos
105 Pos
105 Pos
5
6
5
10 -10 Pos
10 Pos
105 Pos
6
6
6
1.1 x 10
10 Pos
10 Pos
106 Pos
6
6
10 Pos
10 Pos
106 Pos
*As determined by the standard culture method.
Conclusion
Reproducibility among personnel was calculated. In only one of 12 samples (each in duplicate) there was a
disagreement between technicians’ results. Seventy (70) of 72 determinations exhibited complete
agreement between technicians’ results (70/72 x 100 = 97.2% agreement). The results correlated well with
the concentration of bacteria determined by culture.
BIBLIOGRAPHY
1.
2.
3.
4.
5.
Rosenberg, M., S.A. Berger, M. Barki, S. Goldberg, A. Fink and A. Miskin. 1992. Initial Testing of a Novel urine
Culture Device. Journal of Clinical Microbiology. p. 2686-2691.
The Oxoid Manual, 7th Edition. 1995. Oxoid Unipath, Basingstoke, England. 2-75: 2-76
The Oxoid Manual, 7th Edition. 1995. Oxoid Unipath, Basingstoke, England. 2-144:2-145.
Degre, M. 1970. Quantitative and semiquantitative methods for detection of bacteriuria. Translated from Tidsskrift
for Den norske laegeforening 90: 27-30.
McAllister, T.A. 1973. The day of the Dipslide. Nephron 11:123-133.
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6.
Mackey, J.P. and G.H. Sandys. 1965. Laboratory diagnosis of infections of the urinary tract in general practice by
means of a dip-inoculum transport medium. British Medical Journal. 2:1286-1288.
7. Kass, E.H. 1957. Bacteriuria and the diagnosis of infections of the urinary tract. Arch. Int. Med. 100:709-714.
8. Berger, S.A., B. Bogokowsky, and C. Block. 1990. Rapid screening of urine for bacteria and cells by using a
catalase reagent. J. Clin. Micro. 28:1066-1067.
9. Clarridge, J.E., M.T. Pezzlo, and K.L. Vosti. 1987. Laboratory diagnosis of urinary tract infections. In Cumitech 2A.
American Society for Microbiology, Washington D.C.
10. Isenberg, H.D. and R.F. D’Amaato. 1985. Indigenous and pathogenic microorganisms of humans, p. 30-31. In E.H.
Lennette, A. Ballows, W.J. Hausler, Jr., and H.J. Shadomy, Manual of Clinical Microbiology, Washington, D.C.
Manufactured by:
Novamed Ltd.
28, Pierre Koenig Street
Talpiot Industrial Area
POB 53231 Jerusalem 91531
ISRAEL
Tel.: +972.2 6781861
Fax: +972.2 6781852
E-mail: [email protected]
Ver. 2003.1 Printed June 2003
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Novamed Ltd.
DIASLIDE Urine Culture Device
URIselect 3 / MacConkey Agar
Catalog No. DS-105
INTENDED USE
Diaslide Urine Culture Device (UCD) is a semi-quantitative screening culture device for detecting,
enumerating and identifying specific bacteria in urine1. The device is intended for use in physician’s office
laboratories (POL) and clinical laboratories as an aid in the diagnosis of urinary tract infection (UTI).
SUMMARY AND EXPLANATION
DIASLIDE URIselect 3 / MacConkey Agar UCD consists of a transparent, hinged plastic casing containing
face-to-face plates of URIselect 3 and MacConkey Agars with a plastic sampler having two bent tips located
between.
URIselect 3 Agar
Non-selective enriched medium allowing the growth of all UTI pathogens, even those with special nutritional
requirements such as group B Streptococci. The medium composition restricts swarming phenomenon of
Proteus spp. assuring better isolation in mixed cultures. The chromogenic and chemical substrates in the
agar yield to the direct differentiation of E. coli -Klebsiella - Enterobacter - Serratia (KES), and Proteus Morganella - Providencia (PMP)-groups and Enterococci. A specificity of URIselect 3 medium is to allow for
the detection of Staphylococcus saprophyticus, which produces light-rose colored colonies. The
differentiation between the different bacterial species or genus is achieved by:
 Chromogenic substrates detecting enzymatic activity of the lactose metabolism, which is split with
the release of a pink to red dye.
 A chromogenic substrate detecting the activity of Beta-glucosidase, which is split with the release
of a blue-green dye. The respective colonies are stained red, (E. coli), metallic-blue, ( KES,
Citrobacter) or blue-green, (Enterococci, S. agalactiae).
 Tryptophan for the detection of tryptophan deaminase, (TDA) of Proteus-Morganella-Providencia
(PMP) group, and for Indole spot tests. The PMP Beta-glucosidase-negative strains produce clear
colonies with a diffusible beige-brown pigment and PMP Beta-glucosidase-positive strains produce
bluish colonies with a slight brown background.
MacConkey AGAR
A selective medium, enabling the differentiation between coliforms and non-lactose fermenters with
inhibition of Gram-positive micrococci3. Most urinary tract pathogens grow on it, whereas most
contaminants are inhibited. Current routine methods for bacteriological examination of urine are the classic
Petri dish culture method4 and the dipslide technique5. Diaslide UCD combines the advantages of both
techniques, enabling bacterial enumeration and isolation following a simple, user-friendly procedure.
PRINCIPLES OF THE PROCEDURE
The two bent tips of the sampler are dipped into the urine specimen and take up a standard volume of
urine. The sampler is then pulled out through the casing, simultaneously inoculating the surfaces of both
media with a streaking dilution. As a consequence, the number of bacteria deposited on the media is in
direct proportion to the number of bacteria present in the specimen6. Following incubation, the number of
bacterial colonies cultures is compared with the Colony Density Chart to determine the level of bacteria in
the urine sample.
KIT CONTENTS
DIASLIDE URIselect 3 / MacConkey Agar UCD
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Kovac’s reagent )1 plastic dropper bottle containing 5 ml of reagent) is provided with 500 pcs of
DIASLIDE URIselect 3 / MacConkey Agar UCD.
MATERIAL REQUIRED BUT NOT PROVIDED
Incubator (37  1C)
Incubation Stand
WARNING AND PRECAUTIONS
1. For In Vitro Diagnostic Use.
2. Use aseptic technique and established laboratory procedure in handling
material.
3. Do not incubate in CO2
and disposing of infectious
STORAGE
1. Store DIASLIDE URIselect 3 / MacConkey Agar UCD at 2-8C.
2. Protect contents from direct light to ensure product stability through the expiration date.
3. Upon opening the bag, store unused Diaslide UCD units by rolling the open end of the bag and sealing
it tightly with tape over the entire length of the opening.
EXPIRATION DATE
1. The expiration date applies to the product in its intact container when stored as directed.
2. Do not use Diaslide UCD exhibiting any of the following characteristics: discoloration, dehydration,
wrinkling or shrinkage of the agar surface; microbial growth prior to inoculation.
3. Avoid use of UCD if an atypical cultural growth occurs during Quality Control procedures.
SPECIMEN COLLECTION
Clean the genital area and collect a midstream urine specimen in a clean container. Inoculate the urine as
soon as possible following collection. If storage of the urine specimen is necessary, maintain the specimen
at 4C in a closed sterile container. Storage time should not exceed two hours.
PROCEDURE
1. Remove Diaslide UCD from its packaging, being careful not to touch the sampler tips.
2. Dip the sampler tips into the thoroughly mixed urine sample, making sure that both tips are immersed up
to the point where they meet.
3. Holding Diaslide UCD vertically with one hand, use the other hand to draw the sampler up through the
casing in a straight manner. Discard the sampler.
4. Write patient ID directly on the Diaslide casing, or apply an in-house ID label, as required.
5. Return the Diaslide casing into its packaging, close firmly and stand upright in the incubation stand.
6. Incubate at 35C 2º for 18-24 hours, using aerobic conditions.
7. It is recommended to incubate, in a vertical position using the incubation stand.
Fig. 1
Fig. 2
Fig. 3
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RESULTS
Following incubation, examine Diaslide UCD for bacterial growth, which may be evidenced by visible
colonies on the agar surface. Since each colony results from growth of a single bacterial cell, and since the
sampler takes up a standard volume of urine, the numbers of colonies can indicate the “colony count” of the
specimen, the approximate number of bacteria per ml (CFU/ml) of urine. Culture-negative samples can be
observed without opening the Diaslide casing. If microbial growth is present , open the Diaslide UCD
casing and match the “colony density” on the agar surface with the printed illustration it most closely
resembles on the Colony Density Chart (See below).
TABLE 1: The relationship between bacterial concentration (CFU/ml) and the approximate colony
count of the Colony Density Chart.
ORGANISM
CFU/ml
MacConkey Agar
103
4
104
15
Escherichia coli
105
100
6
10
-400
INTERPRETATION OF RESULTS
Bacterial count
As a rule, tests yielding 105 CFU/ml are regarded as positive (significant level of organism), 104 CFU/ml
as negative, and between 104 and 105 CFU/ml as borderline, which calls for a repeat assessment7.
Symptomatic patients having colony counts less than 105 CFU/ml require
evaluation based on clinical information. 8, 9 Many factors, such as use of antimicrobial therapy, time of urine
incubation in the bladder (e.g., first voided urine), and proper specimen collection, may influence the colony
count obtained. In all cases, the physician must be the final judge of the proper interpretation of Diaslide
UCD test results.
COLONY DENSITY CHART
Colony Morphology & Principle of Identification with URISelect 3 Agar
Presumptive identification is based on typical morphology and colony color. Chromogenic URISelect 3 agar
enables identification of bacteria by distinct color differences among different types of organisms.
E.coli can be identified based on demonstration of the activity of two enzymes, β-glucuronidase and βtryptophanase. β-glucuronidase cleaves the first chromogenic substrate contained in the medium, causing
the colonies to turn pink. Proteus is characterized by tryptophan deaminase activity; Proteus mirabilis is
indole-negative. Enterococci produce a β- glucosidase (esculinase), which cleaves the second chromogenic
substrate contained in the medium, causing the colonies to turn turquoise blue.
Revelation of the Various Enzyme Activities
1. Spontaneous
For β-glucuronidase and β-glucosidase: change in color of the colonies after incubation at 37°C for
18 to 24 hours.
Positive reaction of β-galactosidase activity – pink color of colonies.
β-glucuronidase activity suggesting presence of E. coli, to be confirmed by testing for indole production:
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

indole production indicates on E. coli:
absence of indole production indicates a need for identification using conventional methods.
Positive reaction of β- glucosidase activity – turquoise blue color of colonies: examine the culture
under a microscope:
 COCCI, with small colonies (0.5 to 1.0 mm in diameter) and a bright, frank blue color are
Enterococcus species or group B Streptococci. If any of these characteristics is lacking, identify
the organism using conventional methods.
 BACILLI, with large colonies (2.0 to 3.0 mm in diameter) and a blue or greenish-blue color are
probably KES group organisms (Klebsiella, Enterobacter, Serratia or Citrobacter species) and
should be identified using a conventional method.
Negative reaction: No coloration.
2. Spontaneous or after addition of a reagent:
Tryptophan deaminase activity (TDA) indicating an organism of the Proteus-Providencia-Morganella group.
Positive reaction of TDA - spontaneously, orange-brown color of the colony, with or without a change on
agar color to brown. If the change in color is slight, revelation of the enzyme can be enhanced by
depositing a drop of iron perchlorite (FeCl3) onto a colony: the reagent turns brown-green within a few
seconds. Test for indole production: for cultures containing only one kind of organism, deposit one drop of
Kovac’s reagent directly onto a brown colony:
 if the color of the reagent does not change within 15 seconds, there is no indole production
indicating a Proteus mirabilis;
 if the reagent turns pink within 15 seconds there is indole production indicating an indole+
Proteus, a Providencia, or a Morganella. Precise identification should be performed using a
conventional method.
Negative reaction: No coloration on an isolated colony.
Detection of indole production:
For cultures containing only one kind of organism, deposit one drop of Kovac’s reagent on a well-isolated
suspect-colony, directly onto the agar:
 if the reagent turns pink within no more than 15 seconds the organism is indole positive;
 if the reagent is still colorless after 15 seconds, the organism is indole negative.
TABLE 2: The relationship between bacterial enzyme activities and the colony morphology on
chromogenic URISelect 3 agar
Organism
E. coli
KES group
organisms
Proteus mirabilis
Proteus vulgaris,
Morganella,
Providencia
Enterococcus
faecalis
Group B
Streptococcus
Staphylococcus
Colonies morphology
Enzyme activities
Size(Ø mm)
Color
β-glucuronidase ß-glucosidase
2.0 or 3.0
Pink
+
Turquoise blue
+
2.0 or 3.0
or blue purple
2.0
Orange brown
2.0
0.5 – 1.0
0.5 – 1.0
1.0 or 2.0
Orange brown
Bright, frank,
blue
Bright, frank,
blue
White
50
Indole
+
TDA
-
+/-
-
-
+
-
-
+
+
-
+
-
-
-
+
-
-
-
-
-
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TABLE 3: Interpretation of DIASLIDE URIselect 3 / MacConkey Agar UCD results
CHROMOGENIC URISelect 3 AGAR SIDE
POSITIVE
NEGATIVE
GROWTH
NO GROWTH
105 or more CFU / ml*
Less than 105 CFU / ml*
Two or less types of
microorganisms
More than two types of
microorganisms
CHROMOGENIC SIDE
MACCONKEY SIDE
Pink colonies. Confirm with a positive
reaction of the colony with the Kovac’s
Pink-red colonies
Reagent (the reagent turns pink within 15
sec.).
Turquoise blue or blue purple large
Pink-red colonies
colonies
Orange brown color of the colony, with or
Translucent colonies
without a brown color of the agar
Spreading and flat yellowish-green colonies
Clear colonies
with metallic sheen
Small or extremely small (0.5 to 1.0 mm in
No Growth
diameter) bright, frank, blue colonies
No Growth
White colonies
*Each individual laboratory should determine its own cutoff point
RESULTS
E.coli
Klebsiella, Enterobacter, Serratia
and Citrobacter species
Proteus, Providencia and Morganella
species
Pseudomonas species
Enterococcus or group B Streptococcus
species
Staphylococcus species
Presumptive identification is based on typical morphology and colony color (Table 2).
TABLE 2A. Typical colonial morphology on Diaslide MacConkey Agar
ORGANISM
MacConkey Agar
Escherichia coli
Round, pink-red, medium size
Round, red colored, medium size
E. aerogenes
Dark-pink, mucoid, large
Klebsiella pneumoniae
Colorless, translucent, medium size
Proteus mirabilis
Pink, flat, large
Pseudomonas aeruginosa
No growth
Streptococcus faecalis
No growth
Streptococcus agalactiae
No growth
Staphylococcus aureus
No growth
Streptococcus saprophyticus
No growth
Candida spp.
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TABLE 2B. Typical Cultural Response on Colorex® Orientation Agar
ORGANISM
*Escherichia coli
E. aerogenes
Klebsiella pneumoniae
Proteus mirabilis
Pseudomonas aeruginosa
Streptococcus faecalis
Streptococcus agalactiae
Staphylococcus aureus
Streptococcus saprophyticus
Candida spp.
URIselect 3 Agar
Pink-red, flat, medium size
Metallic blue, mucoid, medium size
Metallic blue, mucoid, rather big
Beige to brown on beige background, medium size
Creamy, green, non-swarming, rather big
Turquoise, small
Translucent, light blue shadowed, small
White, compound, medium size
Pink-light purple convex compound, opaque, small
Creamy, wet, small
* Confirm for complete identification by Positive Indole spot test with Kovac’s reagent, yielded pink color.
Bibliography
1. Rosenberg, M., S.A. Berger, M. Barki, S. Goldberg, A. Fink and A. Miskin. 1992. Initial Testing of a
Novel urine Culture Device. Journal of Clinical Microbiology. p. 2686-2691.
2. The Oxoid Manual, 7th Edition. 1995. Oxoid Unipath, Basingstoke, England. 2-144:2-145.
3. Degre, M. 1970. Quantitative and semiquantitative methods for detection of bacteriuria. Translated from
Tidsskrift for Den norske laegeforening 90: 27-30.
4. McAllister, T.A. 1973. The day of the Dipslide. Nephron 11:123-133.
5. Mackey, J.P. and G.H. Sandys. 1965. Laboratory diagnosis of infections of the urinary tract in general
practice by means of a dip-inoculum transport medium. British Medical Journal. 2:1286-1288.
6. Kass, E.H. 1957. Bacteriuria and the diagnosis of infections of the urinary tract. Arch. Int. Med. 100:709714.
7. Clarridge, J.E., M.T. Pezzlo, and K.L. Vosti. 1987. Laboratory diagnosis of urinary tract infections. In
Cumitech 2A. American Society for Microbiology, Washington D.C.
8. Isenberg, H.D. and R.F. D’Amaato. 1985. Indigenous and pathogenic microorganisms of humans, p. 3031. In E.H. Lennette, A. Ballows, W.J. Hausler, Jr., and H.J. Shadomy, Manual of Clinical Microbiology,
Washington, D.C.
PACKAGING
Diaslide URIselect 3 / MacConkey Urine Culture Device 10 tests
10 x 10 tests DS-105
Manufactured by:
Novamed Ltd.
28, Pierre Koenig Street
Talpiot Industrial Area
POB 53231 Jerusalem 91531
ISRAEL
Tel.: +972.2 6781861
Fax: +972.2 6781852
E-mail: [email protected]
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Novamed Ltd.
DIASLIDE Urine Culture Device
TSA Blood / MacConkey Agar
Catalog No: DS-108
INTENDED USE
Diaslide Urine Culture Device (UCD) is a semi-quantitative screening culture device for detecting,
enumerating and identifying specific bacteria in urine1. The device is intended for use in physician’s
office laboratories (POL) and clinical laboratories as an aid in the diagnosis of urinary tract infection
(UTI).
SUMMARY AND EXPLANATION
Diaslide TSA Blood / MacConkey Agar UCD consists of a transparent, hinged plastic casing
containing face-to-face plates of TSA with sheep blood and MacConkey agars with a plastic sampler
having two bent tips located between.
TRYPTONE SOYA AGAR2
A general purpose medium for the growth of a wide variety of organisms, and determination of
haemolysis. The agar and medium containing two peptones and sheep blood and is suitable for the
cultivation of aerobes and anaerobes and for determining hemolytic reactions.
MacConkey AGAR
A selective medium, enabling the differentiation between coliforms and non-lactose fermenters with
inhibition of Gram-positive micrococci3. Most urinary tract pathogens grow on it, whereas most
contaminants are inhibited. Current routine methods for bacteriological examination of urine are the
classic Petri dish culture method4 and the dipslide technique5. Diaslide UCD combines the advantages
of both techniques, enabling bacterial enumeration and isolation following a simple, user-friendly
procedure.
PRINCIPLES OF THE PROCEDURE
The two bent tips of the sampler are dipped into the urine specimen and take up a standard volume of
urine. The sampler is then pulled out through the casing, simultaneously inoculating the surfaces of both
media with a streaking dilution. As a consequence, the number of bacteria deposited on the media is in
direct proportion to the number of bacteria present in the specimen6. Following incubation, the number
of bacterial colonies cultures is compared with the Colony Density Chart to determine the level of
bacteria in the urine sample.
KIT CONTENTS
Tryptone Soya Agar
Formula per liter
Tryptone …………………..
Soya Peptone……………..
Sodium Chloride………….
Agar………………………...
pH 7.30.2
gr/litre
MacConkey Agar
Formula per liter
Peptone............................
Lactose.............................
Bile salts No. 3.................
Sodium Chloride...............
Agar..................................
Neutral Red......................
Crystal Violet....................
Final pH 7.2  0.2 at 25C
15g
5g
5g
15g
gr/litre
20 g
10 g
1.5 g
5g
15 g
0.03 g
0.001 g
MATERIAL REQUIRED BUT NOT PROVIDED
Incubator (37  1C)
Incubation Stand
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WARNING AND PRECAUTIONS
1. For In Vitro Diagnostic Use.
2. Use aseptic technique and established laboratory procedure in handling and disposing of infectious
material.
3. Do not incubate in CO2
STORAGE
1. Store Diaslide UCD at 2-8C.
2. Protect contents from direct light to ensure product stability through the expiration date.
3. Upon opening the bag, store unused Diaslide UCD units by rolling the open end of the bag and
sealing it tightly with tape over the entire length of the opening.
EXPIRATION DATE
1. The expiration date applies to the product in its intact container when stored as directed.
2. Do not use Diaslide UCD exhibiting any of the following characteristics: discoloration,
dehydration, wrinkling or shrinkage of the agar surface; microbial growth prior to inoculation.
3. Avoid use of UCD if an atypical cultural growth occurs during Quality Control procedures.
SPECIMEN COLLECTION
Clean the genital area and collect a midstream urine specimen in a clean container. Inoculate the urine
as soon as possible following collection. If storage of the urine specimen is necessary, maintain the
specimen at 4C in a closed sterile container. Storage time should not exceed two hours.
PROCEDURE
1. Remove Diaslide UCD from its packaging, being careful not to touch the sampler tips.
2. Dip the sampler tips into the thoroughly mixed urine sample, making sure that both tips are immersed
up to the point where they meet.
3. Holding Diaslide UCD vertically with one hand, use the other hand to draw the sampler up through
the casing in a straight manner.
Discard the sampler.
4. Write patient ID directly on the Diaslide casing, or apply an in-house ID label, as required.
5. Return the Diaslide casing into its packaging, close firmly and stand upright in the incubation
stand.
6. Incubate at 35C for 18-24 hours, using aerobical conditions.
7. It is recommended to incubate, in a vertical position using the incubation stand.
RESULTS
Following incubation, examine Diaslide UCD for bacterial growth, which may be evidenced by visible
colonies on the agar surface. Since each colony results from growth of a single bacterial cell, and since
the sampler takes up a standard volume of urine, the numbers of colonies can indicate the “colony
count” of the specimen, the approximate number of
bacteria per ml (CFU/ml) of urine. Culture-negative samples can be observed without opening the
Diaslide casing. If microbial growth is present, open the Diaslide UCD casing and match the “colony
density” on the agar surface with the printed illustration it most closely resembles on the Colony Density
Chart (See below).
TABLE 1: The relationship between bacterial concentration (CFU/ml) and the approximate colony count
of the Colony Density Chart.
ORGANISM
Escherichia coli
CFU/ml
103
MacConkey Agar
4
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104
105
106
15
100
-400
INTERPRETATION OF RESULTS
Bacterial count
As a rule, tests yielding 105 CFU/ml are regarded as positive (significant level of organism), 104
CFU/ml as negative, and between 104 and 105 CFU/ml as borderline, which calls for a repeat
assessment7. Symptomatic patients having colony counts less than 105 CFU/ml require
evaluation based on clinical information8, 9 . Many factors, such as use of antimicrobial therapy, time of
urine incubation in the bladder (e.g., first voided urine), and proper specimen collection, may influence
the colony count obtained. In all cases, the physician must be the final judge of the proper interpretation
of Diaslide UCD test results.
Colony Morphology
Presumptive identification is based on typical morphology and colony color.
TABLE 2. Typical colonial morphology on Diaslide UCD media
ORGANISM
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus mirabilis
Proteus Vulgaris
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus faecalis
MacConkey Agar
Pink-red
Pink-red
Clear
Translucent
Clear
No growth
No growth
No growth
Typical Cultural Response on TSA Blood Agar
Organism
Escherichia coli
Staphylococcus aureus
Streptoccoccus pyogenes
Streptoccoccus pneumoniae
Growth
excellent
excellent
excellent
excellent
Hemolysis
none
beta/gamma
beta
alpha
Since other organisms also grow on these media, full biochemical analysis and antimicrobial
susceptibility testing are required for complete identification.
BIBLIOGRAPHY
1.
Rosenberg, M., S.A. Berger, M. Barki, S. Goldberg, A. Fink and A. Miskin. 1992. Initial Testing of
a Novel urine Culture Device. Journal of Clinical Microbiology. p. 2686-2691.
2.
The Oxoid Manual, 7th Edition. 1995. Oxoid Unipath, Basingstoke, England. 2-216.
3.
The Oxoid Manual, 7th Edition. 1995. Oxoid Unipath, Basingstoke, England. 2-144:2-145.
4.
Degre, M. 1970. Quantitative and semiquantitative methods for detection of bacteriuria.
Translated from Tidsskrift for Den norske laegeforening 90: 27-30.
5.
McAllister, T.A. 1973. The day of the Dipslide. Nephron 11:123-133.
6.
Mackey, J.P. and G.H. Sandys. 1965. Laboratory diagnosis of infections of the urinary tract in
general practice by means of a dip-inoculum transport medium. British Medical Journal. 2:12861288.
55
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7.
8.
9.
Kass, E.H. 1957. Bacteriuria and the diagnosis of infections of the urinary tract. Arch. Int. Med.
100:709-714.
Clarridge, J.E., M.T. Pezzlo, and K.L. Vosti. 1987. Laboratory diagnosis of urinary tract
infections. In Cumitech 2A. American Society for Microbiology, Washington D.C.
Isenberg, H.D. and R.F. D’Amaato. 1985. Indigenous and pathogenic microorganisms of
humans, p. 30-31. In E.H. Lennette, A. Ballows, W.J. Hausler, Jr., and H.J. Shadomy, Manual of
Clinical Microbiology, Washington, D.C.
Manufactured by:
Novamed Ltd.
28, Pierre Koenig Street
Talpiot Industrial Area
POB 53231 Jerusalem 91531
ISRAEL
Tel.: +972.2 6781861
Fax: +972.2 6781852
E-mail: [email protected]
56
03-04/02