INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY

MED-21A- Mr. Ariel Salvacion

• An example of a simple microscope is a

magnifying lens that enlarges objects that
THE MICROSCOPE are difficult to view with the unaided eye.
• Movie theater projection units incorporate
this system efficiently

CARE AND USE OF THE MICROSCOPE

• Microscopes available today reflect

improvement in every aspect from the first
microscope of Anton van Leeuwenhoek
(1632-1723).
• Advanced technology as applied to
microscopy has resulted in computer
designed lens systems, sturdier stands,
perfected condensers, and built-in
illumination systems.
• Microscopes can be fitted with multiple
viewing heads for teaching or conferences,
or they can be attached to a computer to
allow an object to be projected onto a
monitor or a large screen.
• Regular care and proper cleaning ensure
continued service from this powerful
diagnostic instrument.
• The references listed at the end of this
chapter address the physical laws of light
and illumination as applied to microscopy

PRINCIPLES OF MICROSCOPY
• In the compound microscope, a magnified
intermediate image of the illuminated
specimen is formed in the optical tube by
each objective lens.
• This image is then magnified again and
viewed through the eyepiece as an enlarged
virtual image that appears to be located
about 10 inches from the eye.
• Microscopists must focus their eyes in that Compound microscope
more distant plane, rather than trying to • Two separate lens systems are used
focus at the distance of the microscope (objective and eyepiece).
stage.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

• Each objective lens forms a magnified image 4. The neck, or arm— provides a structural
of the illuminated specimen in the optical site of attachment for the revolving
tube. nosepiece.
• Eyepiece lenses further magnify this image 5. The stand— is the main vertical support of
so that the microscopist sees an enlarged the microscope. The stage assembly,
virtual image that appears to be together with the condenser and base, is
approximately 10 inches from the eye. supported by the stand.
• It employs two separate lens systems; 6. The revolving nosepiece holds the
objective and eyepiece, the product of objectives and allows for easy rotation from
which produces the final magnification. one objective lens to another.
• Standard microscopes use bright-field - The working distance (WD) between the
illumination in which light passes through objectives and the slide varies with the
the thin specimen make and model of the microscope.
• There are usually three or four objective
COMPONENT PARTS AND THEIR FUNCTIONS lenses , each with a specific power of
1. The eyepieces or oculars - usually are magnification.
equipped with 103 lenses (degree of • Engraved on the barrel of each objective
magnification is 103). lens is the power of magnification and the
- The lenses magnify the intermediate numerical aperture (NA).
image formed by the objective lenses in • The NA is related to the angle of light
the optical tube; they also limit the area collected by the objective; in essence, it
of visibility. indicates the light gathering ability of the
- may have either one or two adjustable objective lens.
eyepieces. ➢ Functionally, the larger the NA, the
- All eyepieces should be used correctly greater the resolution or the ability
for optimal focus. to distinguish between fine details
- Eyepieces should not be interchanged of two closely situated objects.
with the eyepieces of the same model • Four standard powers of magnification and
or other models of microscopes, NA used in the hematology laboratory are;
because the eyepieces in a pair are Low power 103/0.25
optically matched. High power, dry 403/0.65 or 453/0.66
2. The interpupillary control is used to adjust
OI (oil Immersion) 503/0.90
the lateral separation of the eyepieces for
OI 1003/ 1.25
each individual. When it is properly ▪ The smaller the magnification, the
adjusted, the user should be able to focus larger the viewing field; the larger the
both eyes comfortably on the specimen and magnification, the smaller the viewing
visualize one clear image. field.
3. The optical tube connects the eyepieces ▪ Total magnification= magnification of
with the objective lens. The intermediate the eyepiece X magnification of the
image is formed in this component. The objective lens. (eyepiece X objective
standard length is 160 mm, which,
lens)
functionally, is the distance from the real
image plane (eyepieces) to the objective
lenses.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

o Example: 103 (eyepiece) X 7. The stage supports the prepared

1003 (oil immersion) is 10003 microscope slide to be reviewed. A spring
total magnification. assembly secures the slide to the stage.
• Microscopes employed in the clinical 8. The focus controls (or adjustments) can be
laboratory are used with achromatic or plan incorporated into one knob or can be two
achromatic objective lenses, whose function separate controls.
is to correct for chromatic and spheric - When a single knob is used, moving it
aberrations. in one direction engages the coarse
• Chromatic aberrations are caused by the control, whereas moving it in the
spheric surface of the lens, which acts as a opposite direction engages the fine
prism. As the various wavelengths pass control.
through the lens, each focuses at a different - One gradation interval of turning is
point, which gives rise to concentric rings of equivalent to 2 mm.
color near the periphery of the lens. - Many microscopes are equipped with
• Spheric aberrations result as light waves two separate adjustments: one coarse
travel through the varying thicknesses of and one fine. The order of usage is the
the lens, blurring the image. The achromatic same: engage the coarse adjustment
objective lens brings light of two colors into first and then fine-tune with the fine
focus, partially correcting for the adjustment.
aberrations. When achromatic objective 9. The condenser— consist of several lenses in
lenses are used, the center of the field is in a unit
focus, whereas the periphery is not. - may be permanently mounted or
• A plan achromatic lens provides additional vertically adjustable with a rack-and-
corrections for curvature of the field, which pinion mechanism.
results in a flat field with uniform focus. - It gathers, organizes, and directs the
• Plan achromatic lenses sometimes are light through the specimen.
referred to as flat field lenses. - Attached to and at the bottom of the
• Critical microscopy applications may require condenser is the aperture diaphragm,
a plan apochromatic lens, which brings light an adjustable iris containing numerous
of three colors into focus and almost leaves that control the angle and
completely corrects for chromatic amount of the light sent through the
aberration. specimen.
- This type of objective lens is more - The angle, also expressed as an NA,
expensive and is rarely needed for regulates the balance between contrast
routine laboratory use. (ability to enhance parts within a cell)
• A set of lenses with corresponding focal and resolution (ability to differentiate
points all in the same plane is said to be fine details of two closely situated
parfocal. objects).
- As the nosepiece is rotated from one - The best resolution is achieved when
magnification to another, the specimen the iris is used fully open, but there is
remains in focus, and only minimal fine some sacrifice of image contrast.
adjustment is necessary - In practice, this iris is closed only
enough to create a slight increase in

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

image contrast. Closing it beyond this Tungsten possesses a high melting point
point leads to a loss of resolution. and gives off bright yellowish light. A blue
- The stage and condenser consist of a (daylight) filter should be used to eliminate
swing-out lens, an aperture diaphragm, the yellow color produced by tungsten. The
a control for vertical adjustment of the rheostat or light control knob or lever turns
condenser, and two centering screws on the light and should be used to regulate
for adjustment of the condenser. the brightness of the light needed to
- The condenser top lens can swing out of visualize the specimen. The aperture
position. diaphragm control lever should never be
- The stage controls located under the used for this purpose, because closing it
stage move it along an x- or a y-axis. reduces resolving ability.
10. The field diaphragm is located below the
condenser within the base. OPERATING PROCEDURE WITH KOEHLER
- When it is open, it allows a maximally ILLUMINATION
sized circle of light to illuminate the The procedure outlined here applies to
slide. microscopes with a nonfixed condenser. The
- Almost closing the diaphragm, when following steps should be performed at the start
low power is used, assists in centering of each laboratory session in which the oil
the condenser apparatus by the use of objectives will be used:
two centering screws. 1. Connect the microscope to the power
- Some microscopes have permanently supply.
centered condensers, whereas in others 2. Turn on the light source with the power
the screws are used for this function. switch.
- The glass on top of the field diaphragm 3. Open the condenser aperture and field
protects the diaphragm from dust and diaphragms.
mechanical damage. 4. Revolve the nosepiece until the 103
• Microscopes depend on electricity as the objective lens is directly above the stage.
primary source for illumination power. 5. Place a stained blood film on the stage and
There are two types of brightfield focus on it, using the fixed eyepiece, while
illumination: (1) critical illumination, in covering the other eye. (Do not simply close
which the light source is focused at the the other eye, because this would
specimen, which results in increased but necessitate adjustment of the pupil when
uneven brightness; and (2) the Koehler (or you focus with the other eyepiece.)
Köhler) system, in which the light source 6. Adjust the interpupillary control so that
and the condenser are properly aligned. The looking through both eyepieces yields one
end result of Koehler illumination is a field clear image.
of evenly distributed brightness across the 7. Using the adjustable eyepiece and covering
specimen. This is especially important when the opposite eye, focus on the specimen.
using the oil objectives or when taking Start with the eyepiece all the way out, and
photomicrographs. Tungsten-halogen light adjust inward. If using two adjustable
bulbs are used most frequently as the eyepieces, focus each individually.
illumination source. They consist of a 8. Raise the condenser to its upper limit.
tungsten filament enclosed in a small quartz
bulb that is filled with a halogen gas.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

9. Focus the field so that the cells become and its chromatin pattern; the cytoplasm
sharp and clear. Concentrate on one cell and its color and texture. The objective lens
and place it in the center of the field. should dip into the oil slightly.
10. Close the field (lower) diaphragm. Look
through the eyepieces. A small circle of light Considerations
should be seen. If the light is not in the 1. When revolving the nosepiece from one
center of the field, center it by using the power to another, rotate it in such a
two centering screws located on the direction that the 103 and 403 objective
condenser. This step is essential, because an lenses never come into contact with the oil
off-center condenser will result in uneven on a slide. If oil inadvertently gets onto the
distribution of light. Adjust the vertical high dry objective, clean the objective
height of the substage condenser so that immediately.
you see a sharp image of the field 2. Parcentric refers to the ability to center a
diaphragm, ringed by a magenta halo. If the cell in question in the microscopic field and
condenser is raised too much, the halo is rotate from one magnification power to
orange; if it is lowered too far, the halo is another while retaining the cell close to the
blue. center of the viewing field. Recentering of
11. 11. Reopen the field diaphragm until it is the cell at each step is minimal. Most
nearly at the edge of the field, and fine-tune laboratory microscopes have this feature.
the centering process. 3. In general, when the 103 and 403 objective
12. Open the field diaphragm slightly until it lenses are used, the light intensity should
just disappears from view. be low. When the 503 and 1003 objective
13. Remove one eyepiece and, while looking lenses are used, increase the intensity of
through the microscope (without the the light by adjusting only the rheostat (light
eyepiece), close the condenser aperture control knob or lever) or by varying neutral
diaphragm completely. Reopen the density filters. Neutral density filters are
condenser aperture diaphragm until the used to reduce the amplitude of light and
leaves just disappear from view. Replace the are available in a variety of densities.
eyepiece. 4. Do not change the position of the
14. Rotate the nosepiece until the 403 objective condenser or the aperture diaphragm
lens is above the slide. Adjust the focus (the control lever to regulate light intensity when
correction should be minimal) and find the viewing specimens with the oil immersion
cell that you had centered. If it is slightly off objectives. The condenser should always be
center, center it again with the stage x-y in its upward position as set during the
control. Note the greater amount of detail Koehler illumination adjustment. The
that you can see. aperture diaphragm may be adjusted to
15. Move the 403 objective out of place. Place a achieve proper contrast of the features of
drop of immersion oil on top of the slide. the specimen being viewed.
Rotate the nosepiece until the 1003 5. After setting the Koehler illumination, when
objective lens is directly above the slide. a new slide is to be examined, always bring
Avoid moving a non–oil immersion objective the specimen into focus with the 103
through the drop of oil. Adjust the focus objective first, and then move to the higher
(the correction should be minimal) and magnifications.
observe the detail of the cell: the nucleus

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

IMMERSION OIL AND TYPES removing the dust may scratch the lens.
• Immersion oil is required to increase the Do not use laboratory wipes or facial
refractive index when either the 503 or the tissue to clean the lenses.
1003 oil immersion objective lens is used. 3. Avoid placing fingers on the lens
The refractive index is the speed at which surface. Fingerprints affect the contrast
light travels in air divided by the speed at and resolution of the image.
which light travels through a substance. This 4. Use solvent sparingly. The use of xylene
oil, which has the same properties as glass, is discouraged, because it contains a
allows the objective lens to collect light carcinogenic component (benzene).
from a wide NA, which provides high Xylene is also a poor cleaning agent,
resolution of detail. leaving an oily film on the lens. Lens
• Three types of immersion oil, differing in cleaner or 70% isopropyl alcohol
viscosity, are employed in the clinical employed sparingly on a cotton
laboratory: applicator stick can be used to clean the
1. Type A has very low viscosity and is objective lenses. Alcohol should be kept
used in fluorescence and darkfield away from the periphery of the lenses,
studies. because alcohol can dissolve the
2. Type B has high viscosity and is used cement and seep into the back side of
in brightfield and standard clinical the lens.
microscopy. In hematology, this oil 5. When fresh oil is added to residual oil
is routinely used. on the 1003 objective lens, there may
3. Type C has very high viscosity and is be loss of contrast. Clean off all residual
used with inclined microscopes with oil first.
long-focus objective lenses and 6. Do not use water to clean lenses. If no
wide condenser gaps. lens cleaner is available, use a clean
• Bubbles in the oil tend to act as prisms and microfiber cloth.
consequently reduce resolution. Bubbles 7. When transporting the microscope,
may be created when oil is applied to the place one hand under the base as
slide. They are caused by lowering the support and one hand firmly around the
objective immediately into the oil. Sweeping arm.
the objective from right to left in the oil • In addition to daily care of the microscope,
eliminates bubbles. semiannual or annual maintenance with
thorough cleaning should be done by a
CARE OF THE MICROSCOPE professional. Microscope professionals may
Care of the microscope involves the following recognize and correct problems with
details: mechanics or optics before they are
1. When not in use for an extended period detected by the microscope user. They can
of time, always cover the microscope to correct problems such as sticking of stage
protect it from dust. controls or incorrect optical alignment that
2. Before use, inspect the component can lead to physical problems like carpal
parts. If dust is found, use an air syringe, tunnel syndrome and headaches.
a camel hair brush, or a soft lint-free
cloth to remove it. Using lens paper BASIC TROUBLESHOOTING
directly on a dirty lens without first

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

Most common problems are related to inability installation of an annular diaphragm in the
to focus. Once the operator has ensured that he condenser, together with a phase-shifting
or she is not trying to obtain a “flat field” using element, creates excellent contrast of a cell
an objective lens that is not plan achromatic, against its surrounding background.
the following checklist can aid in identifying the • The principle of phase contrast is related to
problem: the index of refraction and the thickness of
• Eyepieces Clean? a specimen, which produce differences in
• Securely assembled? the optical path. Light passing through a
• Objective lens transparent specimen travels slightly slower
• Screwed in tightly? than light that is unobstructed. The
• Dry objective free of oil? difference is so small that it is not
• Condenser noticeable to the viewer. When a
• Adjusted to proper height? transparent phase plate is placed into the
• Free of oil? microscope, however, the change in phase
• Slide can be increased to half a wavelength,
• Correct side up? which makes the otherwise transparent
objective visible
• Coverslip
• Correct side of blood film?
• Only one coverslip on slide?
• Free of mounting media?
• Light source • This phase difference produces variation in
• Fingerprints on bulb? light intensity from bright to dark, creating
• Bulb in need of changing? contrast in the image. Often the objects
• Light source aligned correctly? appear to have “haloes” surrounding them.
• In hematology, phase-contrast microscopy is
OTHER MICROSCOPES USED IN THE CLINICAL employed in counting platelets in a
LABORATORY hemacytometer, since they are difficult to
Phase-Contrast Microscope visualize and count using brightfield
• The ability to view a stained specimen by microscopy. It also can be used to view
the use of brightfield microscopy is affected formed elements in unstained urine
by two features: sediments.
1. the ability of the specimen to absorb Polarized Light Microscope
the light hitting it, and • Polarized light microscopy is another
2. the degree to which light waves contrast-enhancing technique used to
traveling through the specimen remain identify substances such as crystals in urine
in phase and other body fluids. With brightfield
microscopy, light vibrates in all directions. If
a polarizer (filter) is placed in the light path,
the light vibrates in only one direction or
• Specimens that are transparent or colorless, plane, which creates polarized light. To
such as unstained cells, are not clearly convert a brightfield microscope to a
visualized with brightfield microscopy. polarizing one, two filters are needed. One
Phase-contrast microscopy, through the filter (the polarizer) is placed below the

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

condenser and allows only light vibrating in This diffracted light is picked up by the
the east-west direction perpendicular to the objective lens and appears as bright detail
light path to pass through the specimen. on a black background. Darkfield
The second filter (the analyzer) is placed microscopy is helpful in microbiology in the
between the objective and the eyepiece identification of spirochetes.
and allows only light vibrating in a north-
south direction to pass to the eyepiece.
When the transmission axes of these two
filters are oriented at right angles, no light Specimen collection and
can pass through the pair to the eyepieces. Bacterial Identification
When polarized light (vibrating in an east-
west direction) passes through an optically
active substance such as a monosodium
urate crystal, however, the light is refracted Basic Principles of Specimen Collection
into two beams, one vibrating in the original 1. Collect the specimen in the acute phase
direction (east-west) and one vibrating in a of the infection and before antibacterial
plane 90 degrees to it (i.e., north-south). antibiotics are administered
The refracted light vibrating in the north- 2. Select the correct anatomic site for
south direction can pass through the second collection of the specimen
filter (the analyzer) and is visible at the 3. Collect the specimen using the proper
eyepiece. The magnified crystal appears technique and supplies with minimal
white against a black background. If a first- contamination from normal biota
order red compensator filter also is placed (normal flora)
in the light path below the stage, the 4. Collect the appropriate quantity of
background becomes pink-red, and the specimen
crystal appears yellow or blue, depending 5. Package the specimen in a container or
on its physical orientation relative to the transport medium designed to maintain
incident light path (east-west). Some the viability of the organisms and avoid
crystals can be specifically identified based the hazards that may result from
on their unique birefringent (doubly leakage
refractive) characteristics when polarizing 6. Label the specimen accurately with the
microscopy is used. specific anatomic site and the patient
Darkfield Microscope information – patient’s name and
• Darkfield microscopy is a contrast- unique identification number
enhancing technique that employs a special 7. Transport the specimen to the
condenser. The condenser sends light up laboratory promptly or store the
toward the specimen in a hollow cone. specimen in a transport medium
Because of the high angle of this cone, none 8. Notify the laboratory in advance if
of the illuminating rays enters the objective unsual pathogens or agents of
lens. Without the specimen in place, the bioterrorism is suspected
field would appear black because of the
absence of light. When the specimen is in Collection Procedure
place, and if fine detail exists in the • Clinical specimens should be collected
specimen, light is diffracted in all directions. in sterile containers except for stool

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

specimens which can be collected in medical personnel supervision of the

clean, leakproof containers process
• Generally, swabs are not recommended • First early morning sputum sample is
for collection because they do not preferred
provide sufficient quantity, are easily • The patient should rinse the mouth with
contaminated and can become dried water and expectorate with the aid of a
out leading to a loss of microorganisms. deep cough directly into the sterile
Swabs are appropriate for specimens container (expectorated sputum)
from the upper respiratory tract (URT), • A single specimen should be adequate for
external ear, eye and genital tract. The detection of bacterial LRT infection
tips of swabs may contain cotton, • If fungal or mycobacterial, infections are
Dacron or calcium alginate. suspected, 3 separate early morning
• Cotton tipped swabs tend to have specimens are appropriate
excessive fatty acids which can be toxic • Specimens collected through aerosol
to certain bacteria induction in which the patient breathes
• Swab collection systems are available appropriate aerosolized droplets of a
that may serve as transport media and solution stimulates a cough reflex (induced
protect the specimen from drying sputum)
• Laboratory should be informed if the
Patient Collected Specimen specimen is expectorated or induced
• In certain situations, patients are asked STOOL
to collect the specimen themselves • Specimen of choice for the detection of
• Medical personnel should provide gastrointestinal pathogens
patients with thorough instructions on • Rectal swab can be submitted as long as
how to collect the sample fecal material is visible on the swab
• The most effective method is to provide • A single specimen that has yielded a
verbal and written instructions negative result is not usually significant to
URINE exclude bacteria or parasite infection
• Clean-catch midstream urine specimen • If bacterial infection, 3 specimens should be
• First morning specimen is preffered collected – one a day for 3 days
because it provides a more • If parasite infection, 3 specimens collected
concentrated sample within 10 days
• Patient collects the specimen following LABELLING
cleansing of the external genitals to • Proper identification of each specimen
reduce the presence of indigenous flora includes a label firmly attached to the
• Personnel who collect catheterized container
specimen should also use the o Name
midstream collection to eliminate o Identification number
organisms carried up the urethra during o Room number
catheterization o Physician
Sputum o Culture site
• Collection of a quality sputum sample o Date of collection
requires thorough patient education and o Time of collection

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

1. Specimens collected for microbiology

Collected Specimens should not pose a safety hazard to the
Sputum individuals who handle them
• Collected for the diagnosis of bacterial 2. Leaking containers and specimens with
pneumonia needles attached should be avoided
• Lower respiratory tract (LRT) specimens 3. All specimens must be transported in
are among the most difficult to collect leakproof secondary containers
adequately because they are 4. Transporting personnel should refuse to
contaminated with oropharyngeal flora. transport specimens without the
They are one of the least clinically protection of a secondary container
relevant specimens received for culture 5. Laboratory personnel must adhere to
• Specimens such as blood or strict safety guidelines as they work
bronchoalveolar lavage (BAL) may be with patient’s specimens by wearing
more accurate in the detecting the proper PPEs and specimens opened
etiological agent (microorganism only in a BSC
causing the disease)
Unacceptable Specimen and Specimen
Rejection
• The microbiology laboratory must establish
and publish the criteria for specimen
rejection. The following are examples of
suboptimal specimens that must be
rejected:
1. The information on the requisition does
Requisitions and Laboratory Safety not match the information on the
Requisitions specimen label. If the patient name or
• The requisition should provide the following source does not match, the specimen
information: should be collected again
o Patient’s name 2. The specimen is not submitted in the
o Patient's age (date of birth) and appropriate transport container or the
gender container is leaking
o Patient's room number or location 3. The quantity of the specimen is
o Physician’s name and address inadequate to perform all tests
o Specific anatomic site requested
o Date and time of specimen 4. The specimen transport time is more
collection than 2 hours and the specimen has not
o Clinical Diagnosis or relevant been preserved
patient history 5. The specimen is received in a fixative
o Antimicrobial agents (if the patient such as formalin ; stools for ova and
is receiving) parasites are exception
o Name of individual transcribing the 6. An anaerobic culture is requested on a
orders specimen in which anaerobes are
Laboratory Safety indigenous

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

7. Microbiology processing of a particular 9. Use sterile leak proof container

specimen results in questionable data 10. Properly label container, not lid
(example Foley catheter tips) 11. Transport in secondary container
8. Specimen is dried up (plastic bag) with marked biohazard
9. More than one specimen from the same symbol
source was submitted from the same 12. Protect requisition from contamination
patient on te same day with the 13. Syringes with needled attached should
exception of blood cultures never be transported
10. One swab was submitted with multiple 14. Deliver to lab within 30 mins of
requests for various organisms collection
11. Gram stain of expectorated sputum 15. For prolonged transit, use special
reveals less than 25 white blood cells preservation or holding media
(WBCs) or polymorphonuclears (PMNs)
and more than 10 epithelial cells per
low-power field (lpf) and mixed
bacterial flora
BACTERIAL IDENTIFICATION
• All rejected specimens must be called to the
person in charge of collecting the specimen
• The laboratory should NEVER discard an
Types of Bacteria
unacceptable specimen before contacting a
There are seven main groups of bacteria,
member of the healthcare team
distinguished by their shapes and the type
• The laboratory must document the situation
of cell wall they possess.
including the reason for rejection of the
specimen
Four of the 7 Types are the following:
• If the physician insists on processing an
1. Gram positive cocci
inadequate specimen, the lab report must 2. Gram negative cocci
include a comment explaining the potential 3. Gram positive bacilli
compromised results
4. Gram negative bacilli

Specimen Collection Guidelines:

Microscopic Observation
1. Obtain during acute phase of infection
• Processing patient specimen begins
(WITHIN 2-3 days for viruses)
with a macroscopic observation
2. Collect before antibiotics are
• The gross appearance of the specimen
administered
may provide useful information to both
3. Sample appropriate site
the microbiologist and the physician
4. Aspirates or tissues are preferred to
• Notifications from the macroscopic
swabs
observation should include the
5. Use swabs with Dacron or polyester tips
following:
& plastic shafts, wood, cotton, and
o Swab or aspirate
calcium alginate may be toxic
o Stool consistency (Formed or
6. For anaerobes are preferred to swabs
liquid)
7. Avoid contamination with
o Blood or mucus present
environmental or normal flora
o Volume of specimen
8. Obtain sufficient quantity

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

o Fluid (clear or cloudy) 1. Simple stains

▪ Areas of blood or mucus must 2. Different stains
be selected for culture and 3. Probe-mediated stains
DIRECT microscopic SIMPLE STAINS
examination • Directed toward coloring the forms and
▪ Anaerobic cultures may be shapes present
indicated if gassy, foul smelling DIFFERENTIAL STAINS
or sulfur granules are present • Directed toward coloring specific
▪ Diagnosis is evident if adult components of the elements present
helminths or tapeworm PROBE-MEDIATED STAINS
proglottids are present in the • Diagnostic antibody or DNA probe-
specimen mediated stains
• Directed specifically at the identification
of an organism

Microscopic Observation
1. Cocci are spherical cells, bacilli are rod
shaped. Bacteria that have thick cell
walls are termed as Gram positive
because of the way they take up the
Gram stain. Those with thin walls are
termed as Gram negative
2. The Gram positive bacteria are bacteria
with thick cell walls containing the
teichoic acid retain the crystal violet –
iodine complex dye after decolorization
and appear as deep blue
3. The Gram negative bacteria are bacteria
with thinner cell walls containing
lipopolysaccharides (LPS) that do not
retain the dye complex

Stains
Stains can be categorized as: GRAM STAIN

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MED-21A- Mr. Ariel Salvacion

Gram stain 3. Place the smear in an upright position in

• Developed by Christian Gran in 1884 a staining rack, allowing the excess
Principle water to drain off and the smear to dry
• The steps in this laboratory procedure 4. Examine the stained smear using the
provide for the crystal violet (hexamethyl-p- low-power objective and select an area
rosaniline chloride) to color all cells and to examine more closely using a 40 to
background material a deep blue and for 60 high-power objective. Suspicious
Gram’s iodine to provide the larger iodine areas are evaluated using the 100 oil
element to replace the smaller chloride in objective of the microscope
the stain molecule Result
• The alcohol – acetone complex decolorizer ▪ Gram-positive bacteria stain dark blue to
damages these thin lipid walls and allows blue-black
the stain complex to wash out ▪ All other elements stain safranin red
• All unstained elements are then ▪ Individual structures absorb a different
counterstained with red by safranin dye amount of safranin. Some have prominent
• The differential ability of the Gram stain staining (string avidity) and others are
makes it useful in microbial taxonomy weakly stained (low avidity)
▪ Always check the quality of the stain before
Application of Gram Stain moving to interpretation
• Used routinely and requested in the clinical ▪ Among the gram negative bacteria, the
microbiology laboratory for the primary enterics have strong avidity and stain bright
microscopic examination of specimens red ; the pseudomonads have less or lower
submitted for bacterial smear and culture avidity
• Ideally suited for specimen types in which ▪ Anaerobic bacteria and other thin walled
bacterial infections are strongly suspected Gram negative organisms such as Borrelia,
• Regularly used to characterize bacteria Legionella and Spirillum sp stain weakly
growing on culture media (either in liquid or
solid medium) Gram Stain Reagents
• Specimens submitted such as cerebrospinal ▪ Primary stain – Crystal Violet
fluid (CSF), sterile fluid, expectorated ▪ Mordant – Gram’s Iodine
sputum or bronchoalveolar lavages (BAL), ▪ Decolorizer – Acetone and Alcohol
wound and exudates are routinely stained ▪ Counterstain – Safranin
directly
Procedure
1. Dry the clinical material (specimen) on
the slide for it not to be washed out
during the Gram’s staining procedure.
Adherence can be improved by gently
warming the slide
2. Place the smear on a staining rack and
overlay the surface of the material with
stains in sequence as described in the
previous slide

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INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

▪ 2% acid alcohol (lab prepared)

▪ 1% sulfuric acid (partial acid-fast)
▪ 0.5% aqueous potassium permanganate
▪ 0.3% aqueous methylene blue solution (lab
prepared)
▪ Microscopic slide
▪ Sterial water
Procedure for Fluorochrome Stain
1. Cover samear with TV auramine-
rhodamine T stain and stain for 25
minutes
2. Wash in running tap water
3. Move slides to slide rack on acid alcohol
collection container
4. Flood smears with 0.5% acid alcohol
Acid-fast stain and decolorize for 2 minutes
Principle 5. Wash smears in running tap water
▪ The primary stain binds to mycolic acid in 6. Move slides to original staining rack
the cell walls of the mycobacteria and is 7. Flood smears with potassium
retained after the decolorizing step with permanganate counterstain for 4
acid alcohol. The counterstain does not minutes
penetrate the mycobacteria to affect the 8. Wash smears in running tap water
color of the primary stain 9. Air dry
▪ FOR Nocardia Spp. 10. Examine the smears with the x16 or x40
objectives of the fluorescence
microscope equipped with a filter
Application systems comparable to a BG-12 exciter
▪ The direct smear examination is an filter and an OG-1 filter barrier filter
important diagnostic procedure for the 11. Examine each smear for 3 to 5 minutes
detection of mycobacteria in clinical Result for Fluorescent or Fluorochrome Stain
specimen Fluorescent stain: Mycobacteria stain bright
orange. Count the number of acid-fast bacilli
seen on the smear and report as follows:

Kinyoun Stain
1. Cover the smear with carbolfuchsin and
Material for Fluorochrome Stain stain for 5 minutes
▪ TB auramine-rhodamine stain 2. Wash slides with running tap water
▪ Carbofuchsin stain (either commercially 3. Move slides to staining rack on acid
prepared or laboratory prepared) alcohol collection container
▪ 0.5% acid alcohol (0.5% HCl in 70% ethanol)

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MED-21A- Mr. Ariel Salvacion

4. Decolorize with acid alcohol until no GREEN; if counter stain is Methyline Blue-
more color appears in the washings background is BLUE).
5. Wash slides with running tap water and
move them to original staining rack General Rules for Gram Staining
6. Flood slides with methylene blue • All cocci are gram positive except Neisseria,
counterstain for 1 minute Branhamella, Moraxella, and Velionella.
7. Wash with running water, drain and air • All bacilli are gram negative except
dry Mycobacteria, Clostridium,
8. Examine the smears with oil immersion Corynebacterium, Bacillus, Erysipelothrix,
objective x100 Listeria, Lactobacillus.
Modified Kinyoun Stain (Partial Acid-Fast) • Higher forms of organisms (Actinomyces,
1. Flood the slides with carbofuchsin stain Streptomyces, yeasts, and molds) are gram
for 5 minutes positive.
2. Rinse with running tap water • All spiral organisms are reported as gram
3. Flood slides with 70% ethanol and rinse
with tap water. Repeat until excess red Reason why Gram (+) becomes (-)
dye is removed • Overdecolorization
4. Move slides to rack on acid collection • Smear too thick
container • Improper washing between steps
5. Continuously drop 1% sulfuric acid on • Too old Culture
the smear • Impure or mixed culture
6. Rinse with running tap water Reason why Gram (-) becomes (+)
7. Move slides to original staining rack • Underdecolorization
8. Counterstain with methylene blue for • Smear too thick
30 seconds
Acid-fast Stain
• All bacteria are non-acid fast except
Zeihl–Neelsen Stain (HOT METHOD) Mycobacteria group
1. Cover the smear with a piece of filter • One does not belong to the
paper cut slightly smaller than the slide Mycobacteria group but is slighty acid
2. Layer filter paper with carbofuchsin fast: Nocardia asteroides
stain, with a bunsen burner, heat the
• Acid Fast organism (AFO) are very hard
smears gently until steaming occurs.
to stain due to the presence of
Stain for 5 minutes without additional
unsaponifiable wax called mycolic acid
heating
or hydroxymethoxy acid.
3. Proceed as for Kinyoun stain, beginning
• In staining, the mycolic acid is
with Step 2
temporarily removed through the
steaming process.
Result for Kinyoun stain and Ziehl-Neelsen stain
▪ Mycobacteria stain red
▪ Background material and non-acid-fast
bacteria stain either blue or green.
(Depending on the counterstain used; if
uses Malachite Green- background is

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MED-21A- Mr. Ariel Salvacion

• Diagnostic test for Enteric Bacilli:

1. Growth on Media
Commonly Used Methods of Acid-Fast Staining 2. CHO fermentation test
1. Ziehl -neelsen CHO Fermentation test
2. Kinyoun • TSI (most commonly used) – dispersed in
slant butt
Purpose Ziehl-Neelsen Kinyoun’s Result observation:
Primary Stain Carbol Fuchsin Carbol Fuchsin Alkali pH – red (ALK) yellow (ACID)
H2S production – indicated by
Mordant Heat Phenol, Tergitol blackening
Decolorizer 3% acid alcohol 3% acid alcohol

Counterstain Methylene Blue Malachite

green

Other Staining Methods

1. LANA (L-alanine 4-nitroanilide)
2. 3% KOH method

o To a drop of #% KOH, emulsify a loopful

of bacterial colonies
o Stir continuously for 60 seconds with a
wooden stick
o Loop if gently pulled out

Biochemical Testing
Biochemical Tests
• Are never done in mixed plates
• Pure cultures are used

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MED-21A- Mr. Ariel Salvacion

Gas production - indicated by splitting, peptones in the medium, slant

cracking, bubbling of agar becomes alkaline
• In TSI those organisms that Ferments
sucrose will have an A/A reaction:
o A – yellow
o K – red
o Wherein:
▪ A/A – 2-3 sugars fermented
▪ K/A – glucose fermented
▪ K/K – no sugar fermented

Carbohydrate Fermentation
• Fermentation – utilization of CHO by
bacteria
• In bacteriology, this process is detected
by observing changes in pH indicator as
acid products are formed

Basic Principle of Fermentation Carbohydrate Fermentation

• Glucose is degraded through a series of • Result: Non-Fermenter (NF), Non-Lactose
enzymatic glycolytic cleavage and Fermenter (NLF), Lactose Fermenter (LF)
transformations; the glucose molecule NLF LF LLF
is split into three carbon compounds, • Edwardsiella • Enterobacter • Citrobacter
• Hafnia • Escherichia • Arizona
the most important is pyruvic acid
• Morganella • Klebsiella
• Media used is: TSI (Triple Sugar Ion) –
• Proteus
contains lactose, glucose, sucrose, and • Salmonella
iron • Shigella
o Used to determine whether • Yersinia
gram (-) can ferment lactose, • Serratia
glucose, or sucrose, and forms
H2S
• Bacteria species incapable of fermenting
o Phenol red – pH indicator
glucose cannot utilize lactose.
o If glucose is fermented, but
• Another media that can be used: Klingler’s
becomes yellow, slant is initially
Iron Agar – contains lactose, glucose, and
yellow, since glucose is in low
iron salt
concentration, Organisms under
aerobic conditions uses

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INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion

• C - ability to utilize citrate as a single carbon

H2S Production source
• Indicators: sodium thiosulfate and ferrous
sulfate
• (+) black color or black precipitate

Gas Production
• (+) bubble formation or splitting of the INDOLE TEST
media or complete displacement of the • Based on the ability of organism to produce
media from the bottom of the tube indole from tryptophan
• Media: Tryptophan broth/SIM broth
Beta-galactosidase and ONPG test • Reagent: Ehrlich’s
(Orthonitrophenyl beta-D-galactopyranoside) Reagent/KOVAC’s/Paradimethylbenzaldehyd
• Is a compound structurally similar to lactose e
test that detects the enzyme beta- • Result: (+) pink to wine colored ring at the
galactosidase junction, (-) no color development
• Used to distinguish enteric bacteria • Rapid Spot Indole test (blue) – filter paper
(Salmonella (-), Citrobacter (+)) and to strips impregnated with p-
identify Pseudomonas amminocinamaldehyde reagent, screening
• Orthonitrophenol is a chromophore that is for indole production
released into the medium and detected by a Interpretation Developmen of cherry red color
pale yellow color at the surface of the reagent and broth within
• (+) yellow, (-) no color change seconds after adding KOVAC’s reagent indicates
the presence of indole and the test is positive. If
SIM TEST no color change is observed, then the test is
• Indole production – ability of organism to negative
split tryptophan to form compound indole
• Result (+) pink to wine red colored ring
• Sulfide = (+) blackening of agar = Motility

MEHTYL RED TEST

• Determines the ability of microbes to
IMViC TEST oxidize glucose with producion and
• I – indole production from tryptophan stabilization of high content of acid end
• M – Methyl Red test in which acidification product
of glucose broth due to formation of mixed • Media: MRVP Broth or Clark Lubbs Broth
carboxylic acids from pyruvate • pH indicator: Methyl Red
• Vi – Voges-Proskauer test due to formation
of acetoin from pyruvate in glucose broth

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MED-21A- Mr. Ariel Salvacion

sodium and water to form carbonate, an

alkaline product that raises the pH. The pH
indicator (bromthemol blue) will change the
color of the media, indicating a positive
result.
• Key biochemical property of Salmonella,
Citrobacter, Klebsiella, Enterobacter, and
Serratia

VOGES-PROSKAUER TEST
• Principle: This test determines the
capability of some organisms to produce
non-acidic or neutral end products, such as
acetyl methyl corbinol (acetoin), from the
organic acid that results from glucose
metabolism. This test identifies bacteria
that ferments glucose and leading to 2,3-
butanediol accumulation in the medium.
OXIDASE TEST (CYTOCHROME
• Reagent: alpha napthol and KOH (Barritt’s OXIDASE/INDOPHENOL BLUE)
Medthod) – (+) pink to red color (E. cloacae)
• Principle: This test uses certain reagent dyes
/ (-) no color change (E. coli)
such as p-phenylenediamine
• Reagent: alpha napthol and 40% KOH in dihydrochloride that substitute for oxygen
creatine (Coblentz) – (+) red (S. mutans) / (-) as artificial electron acceptors. It is colorless
yellow (S. minis) in reduced state. In the presence of
cytochrome oxidase and atmospheric
oxygen , p-phenylenediamine is oxidized
forming indophenol blue.
o (+) bluish purple – P. aeruginosa
o (-) no color change – E. coli

CITRATE UTILIZATION TEST

• Principle: Citrate will be the sole carbon URASE TEST
source of the microorganisms Sodium • Principple: This test determines
citrate in the Simmons’s Citrate Agar will be microorganisms that can degrade urea by
the carbon source while the NH4+ as the urease. Urease hydrolyzes urea realising
nitrogen source. When bacteria oxidize ammonia which alkalinizes the medium by
citrate, they remove it from the medium forming ammonium carbonate and CO2.
and liberates CO2. CO2 combines with

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MED-21A- Mr. Ariel Salvacion

o Proteus, Morganella, Providencia – • Determines whether g(-) can decarboxylate

strong urease producers or deaminate lysine and forms H2SS
o Klebsiella – weak urease producers • Media: LIA – contains lysine, peptones,
o Yersinia enterocolitica – frequently a glucose, ferric NH4 citrate and sodium
urease producer thiosulfate
o Media – Christensen’s Urea Agar/Stuart • H2S indicator – ferric ammonium citrate
Urea Broth • Indicator – bromcresol purple
o pH indicator: phenol red • When glucose is fermented, the butt
becomes acidic. (yellow) if decarboxylase is
not produced, the butt remains yellow. If
oxidative deamination of lysine occurs, it
will form a burgundy color on the slant.
Phenyalanine Deaminase Test
• Principle: Some organisms can deaminate
phenylalanine converting it to
phenylpyruvic acid
o (+) dark green color after addition
of ferric chloride used in the initial
differentiation of Proteus,
Providencia, and Morganella
o Media: Phenylalanine agar or Decarboxylase Test (Moeller’s method)
Tryptophan agar • Test measures the ability of an organism to
o Reagent: 10% Ferric chloride decarboxylate an amino acid to form an
o Result: amine
▪ (+) green color on slant (P. • pH indicator – bromcresol purple
vulgaris) • Decarboxylation of amino acids results in
▪ (-) E. coli alkaline pH change
LYSINE DECARBOXYLASE TEST • 3 decarboxylate broths: arginine, lysine, and
• Principle: Some organisms can ornithine
decarboxylate lysine converting it to • It requires acid pH and anaerobic
cadaverine environment
o Media: Lysine Iron Agar/Moeller’s • (+) result = purple alkaline color
agar • Amino acid Decarboxylation
o Lysine deamination is an aerobic o If an Enterobacteriaceae contain
process that occurs on that slant of amino acid decarboxylase, amines
the media. produced by decarboxylase action
o Lysine decarboxylation is an aerobic cause and alkaline pH
process that occurs in the butt of • LOA test
the media o Important in the identification of
Enterobacter, Klebsiella,
Escherichia, and Salmonella

Lysine Iron Agar Test

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MED-21A- Mr. Ariel Salvacion

H2S Production
• Differentiating test for Salmonella and
Shigella
• Systems for H2S detection:
o Lead acetate paper
o SIM tube
o Hektoen and SS agar Mug Test
o XLD agar • Uses 4-methylumbelliferyl-beta-D-
o TSI glucoronide
• (+) – Salmonella, Edwardsiella, Citrobacter, • (+) blue fluorescence – E. coli
Proteus • (-) no fluorescence – P. Aeruginosa
• Result: (+) production of black color

Motility Test
• Ability of an organism to produce proteases
that hydrolyzes gelatin and liquefy solid
gelatin medium.
• Used in the identification of Clostridium, KCN Broth Test
Serratia, Flavobacterium, and Pseudomonas • This test determines whether the microbe
• (+) result gel liquifies / (-) gel solidifies can grow in a medium where potassium
cyanide is present as a carbon and nitrogen
Nitrate Reduction Test source.
• Principle: This test determines • Result (+) tubid; (-) clear
microorganisms that can utilize nitrate as a
terminal electron acceptor during anaerobic
respiration. Nitrate is reduced to nitrite by
nitrate reductase.

Malonate Utilization Test

• Malonate test is a colorimetric test of the
ability of bacteria to use malonate as a
source of carbon, the endpoint of which is

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MED-21A- Mr. Ariel Salvacion

the production of alkaline metabolites that will bind to the surface of the
induce a color change organisms
• Result (+) blue; (-) green • Used to identify bacteria that are difficult to
cultivate in a culture medium
• +Result: Presence of visible clumpls or
agglutination
• Interpretation: Specific antibodies bind to
the bacterial surface antigens, causing the
organism to clump

String Test Particle agglutination

• For identification of Vibrio spp. • Uses artificial carriers, such as latex particles
• Reagent: 0.5% sodium deoxycholate or treated red blood cells, or biological
• Result: (+) string like carriers as reagents, which can absorb test
antigen and react with the specific antibody
present in the patient’s serum
• +Result: Presence of visible clumps or
agglutination
• Examples: MHA-TP, HATTS, and passive
hemagglutination test for streptococci

Immunodiagnosis Latex Agglutination (Antigent Test)

• The antigen in the patient’s speciemen
Seroligical Test/Immunodiagnosis binds to the antibody that is preset on the
• Basic detection of antibody response from surface of the latex beads (reagents)
an antigenic, stimulation either through a • Also used for direct identification of group B
single identification or serial dilution streptococci
• The presence of antibodies implies either a • The advent of the molecular assays limits
pathogen exposure or a subclinical infection the utilization of this method for bacterial
• Rickettsia and spirochetes identification
• Utilized to assist in the detection of
congenital infections in newborn Coagglutination
• TORCH – Toxoplasma gondii, Other • Uses the antibody that is bound to a particle
organisms (Treponema pallidum subsp. to increase the visibility of the agglutination
Pallidum, Parvovirus B19, Varicella-Zoster, reaction between an antigen and an
etc) Rubella virus, Cytomegalovirus, and antibody
Herpes simplex virus • Assist in the detection of N. meningtidis,
Hemophilus, and streptococci
• The antibody (Fc portion) binds to the test-
strain while the test-antigen (suspected
Bacterial Agglutination pathogen) binds to the specific target site in
• Performed by adding antibodies the antibody (Fab portion)
(agglutinins) to the bacterial suspension

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MED-21A- Mr. Ariel Salvacion

• S. aureus organism that contains protein A • Commonly used for the rapid identification
in its cell walls is utilized in this procedure of bacterial and viral antigens in body fluids,
as part of the reagent swabs, and infected cells.
• Monoclonal or polyclonal antibodies, which
are attached to fluorescent dyes, are
Complement Fixation
applied to an antigen that is previously
• Composed of two parts: a test system which treated with either formalin, acetone, or
includes the antigen that causes the disease alcohol, ten the reaction is visualized under
in the patient serum and an indicator the fluorescent microscope where final
system which consists of the sheep's red results can be released within an hour.
blood cells, complement-fixing antibody like • Borrelia, Legionella, Mycoplasma
the immunoglobulin G(IgG), and an pneumonia, Rickettsia, and TORCH
exogenous complement. • Example of fluorescent dyes: Auramine,
• +Result: Absence of lysis with the red blood rhodamine, and fluorescein
cells which means presence of antibody in isothiocyanate(FITC)
the patient serum • +Result: Occurrence of fluorescence as seen
• •-Result: Occurrence of lysis with the red under the microscope
blood cells and the absence of antibodies in • Types: Direct Fluorescent Antibody (DFA)
the test serum Test and Indirect Fluorescent Antibody (IFA)
Test
Flocculation Test
• uses a soluble antigen that reacts with the
antibody in a serum sample. Types of Western Blot
• +Result: Occurrence of macroscopic or Pulsed-field Gel Electrophoresis
microscopic flocculation (precipitation) • Enzyme-digested chromosomal fragments
• Some examples of this test are venereal of bacteria are isolated electrophoretically
disease research laboratory (VDRL) test and in this approach.
rapid plasma reagin (RPR) test. • Can be used in an outbreak investigation.
Bacteriophage Typing
Enzyme-linked Immunosorbent Assay (ELISA) • Based on the specificity of phage surface
• A sensitive and specific method for the receptors for cell surface receptors
detection of antibodies against certain • A bacteriophage is a virus that attacks
pathogens. bacteria.
• This method consist of enzyme-bound Chromatographic Method(Gas Chromatography
antibodies with the antibody-binding sites and High Performance Liquid Chromatography)
free to react with their specific antigen. • Analysis of microbial metabolites, cellular
• Utilized for the diagnosis of infectious fatty acids, and products of pyrolysis of the
diseases such as those caused by Legionella whole bacterial cells.
• Alkaline phosphatase or horseradish • Gas-liquid chromatography is used to detect
peroxidase is used as an enzyme-conjugate the cellular fatty acids of anaerobes.
antibody reagent. • HPLC is utilized for the fatty acid analysis of
• +Result: Colored end product the Nocardia species.
Immunofluorescent Assays

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MED-21A- Mr. Ariel Salvacion

Matrix-Assisted Laser Desorption Ionization- Types of PCR

Time-of-Flight Mass Spectrometry Conventional PCR
• Excellent tool for the rapid identification of • Principle: The DNA sequence is amplified
a wide range of pathogenic species utilizing a Taq polymerase
• Utilized for species, subspecies, and strain • Clinical/Diagnostic Application: Microbial
identification of bacteria. detection, phylogenetic study, gene
• Faster result in terms of bacterial analysis, therapeutic cancer detection,
identification (in mins.) therapeutic management, and DNA profiling
• Components: Ionization Phase and the TOF and cloning
Phase Real-time PCR
• Procedure: bacterial isolate is ionized by • Known as quantitative PCR
transferring it from the culture plate to a • Quantifies the target nucleic acid after each
metal plate, where it is treated with matrix replication cycle in the same PC equipment
solution until it forms a crystallized utilizing commercially available fluorescence
microbial protein or matrix lattice which is detecting thermocyclers.
then analyzed using MALDI-TOF instrument. • Uses fluorescent dyes to mark specific DNA
• This method creates unique mass spectral • Measure the amplified product and
fingerprints that are compared to a massive determine the number of copies of target
database of mass spectra. substance present in the original specimen.
• Advantages: combines amplification and
product detection at one time, used for
multiple sample analysis, reduces cross-
contamination with the amplified
production, and lowers turn-around time
Types of PCR - Real-time PCR
Real-time RT-PCR/Reverse Transcription
Molecular Diagnosis Quantitative PCR
• Considered the most important method for • Useful for measuring the abundance of
microbial identification certain RNAs to determine the gene
• Useful in confirming the taxonomy of the expression.
emerging and re-emerging pathogens. • Gold standard method for the detection of
Polymerase Chain Reaction (Nucleic Acid SARS-CoV-2
Amplification Assay) Multiplex PCR
• Most commonly used amplification • Allows simultaneous amplification of
technique in molecular diagnosis. multiple gene segments rather than
• Increases the nucleic acid of the test sample separate test runs for each.
or target microorganism from a very small
amount to a million copies
• Utilized for rapid detection of nucleic acid in
biological samples, a very significant tool in
the diagnosis of the etiologies of diseases.

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