IMMUNOLOGY & SEROLOGY SECTION

CHAPTER 1: IMMUNOLOGY INITIAL IMMUNE RESPONSE out all the process that macrophages normally
carry out
OVERVIEW OF IMMUNOLOGY ● Once the invading pathogen has broken through the physical
o Neutrophils – phagocytes that circulate in the
● The Immune System is made up of organs, tissues, cells, and and chemical barriers to infection, three things will happen:
blood and can enter the tissues and help by
molecules that all work together to generate an immune 1. Macrophages recognize the pathogen and activate the
destroying invading pathogens.
response that protects humans from microorganisms, innate immune system
● INFLAMMATORY RESPONSE – help to contain and fight
removes toxins, and destroys tumor cells. 2. Dendritic cells pick up antigens from the pathogen and
infection.
● The Immune Response can do the following all within 10 then head off to the blood and lymphatic systems to
o Vasodilation
days: track down the relevant T and B cells of the specific
o Increase vascular permeability
o Identify a threat immune system and activate them.
o Mast Cell degradation – releasing more cytokines
o Mount an attack 3. The invading pathogen activate the complement
that further stimulates the inflammatory response
o Eliminate pathogen system directly via the Lectin pathway and Alternative
o Activation of clotting and kinin system
o Develop mechanism pathway
● ACUTE PHASE RESPONSE – inflammation stimulates
● In some cases, where pathogen is particularly stubborn or if
macrophages and neutrophils to secrete more cytokines
the immune system starts attacking something, Prolonged INNATE IMMUNE SYSTEM notably called interleukins.
Reaction is manifested (months to years) which leads to ● Macrophages recognize the pathogens by specific o Leads to a more systemic inflammatory response
CHRONIC INFLAMMATION characteristics that occur on pathogens but do not occur on o Releases TNF-alpha that is responsible for
● Immune System is categorized into: cells of the body. These characteristics are called Pathogen producing the ff:
o INNATE immune system – Consists of Associated Molecular Patterns (PAMPs) ▪ IL-1 is sent to the brain to produce fever,
components that are already at the location of the ● Macrophages recognize PAMPs using various receptor types reduce appetite, and lethargy.
infection and responds IMMEDIATELY with a including toll-like receptors. ▪ IL-6 is sent to the liver to produce acute
GENERALIZED response ● PHAGOCYTOSIS – the process where macrophages and other phase proteins that act to opsonins.
o SPECIFIC immune system – Involves specialized T phagocytes, like neutrophils, use in order to destroy ▪ IL-8 recruits and activates neutrophils
and B cells that wait in places AWAY from the site pathogens. Once they have recognized the pathogen, they ▪ IL-1 and IL-12 activates Natural Killer cells.
of infection. When activated, they launch a start to wrap their cell membrane around the pathogen and
SPECIALIZED system that fights the specific absorb them within their cell. Pathogens are left inside the
invading pathogen. This TAKES LONGER to phagosome. Phagosomes fuses with surrounding lysozymes
recognize the infection, particularly when a which provide digestive enzymes that break down the
pathogen is invading the body for the first time. pathogen, destroying and processing all components into
harmless waste products.
INVASION OF PATHOGEN o If invading pathogens are harmless enough that
● Initially, there are PHYSICAL BARRIERS that need to be macrophages can deal with them alone, they are
broken before an infection can take place. cleared and goes no further.
o Skin o If attacking army is too many, the macrophages
o Respiratory Tract Mucosa release cytokines (signaling proteins) like local
o Gastrointestinal Tract Mucosa hormones that sounds the alarm of an infection in
o Urinary Tract Mucosa the local area. This leads to a process called
● There are also CHEMICAL BARRIERS that help destroy INFLAMMATION.
● OPSONINS – complex molecules that attach themselves to
harmful pathogens before they can cause any infection such ● CYTOKINES – recruits and activates more cells of the immune
pathogens which makes macrophages and neutrophils to
as the following: system:
recognize and phagocytose to pathogen
o Hydrochloric acid (stomach) o Macrophages
o C-reactive protein (CRP)
o Lysozyme - sweat and tears o Monocytes – precursors to macrophages that
▪ Produced by the liver
o Lactic Acid - vagina float around in the blood. Once they enter tissues,
▪ Response to IL-6
they differentiate into macrophages and can carry
▪ Measures the serum level

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 IMMUNOLOGY AND SEROLOGY SECTION

▪ Marker of inflammation The Spceific Immune Rsponse start in the Dendritic Cells ▪ These proteins float around the blood and
▪ Indication of severity of infection presenting the antigens in their HLA Class 2 molecules to the CD4 attach themselves to antigens that match
cells. These CD-4 cells, which are a type of T-cells, then proliferate their specific variable region on these
SUMMARY: and become T-helper cells. The T-helper cells present antigen in antibodies.
● INNATE IMMUNE SYSTEM is a generalized system that their HLA Class 1 molecule that can be recognized by CD8 cells ▪ It helps the immune system to fight
recognizes and responds to an invading pathogen by: T cells also secrete cytokines that are responsible for making CD8 pathogens in a number of different ways:
o causing an INFLAMMATORY RESPONSE and cells proliferate and differentiate into Cytotoxic T cells. 1. Attach to toxins and neutralizes their
o recruiting CELLS that destroy pathogens by T-helper cells also release cytokines that stimulates B cells to toxic effect
phagocytosis. proliferate and differentiate into plasma cells that can release large 2. attach to receptors of viruses and
quantities of Antibodies, and Memory B cells that hang around as bacteria and prevent them from
COMPLEMENT SYSTEM part of the immune memory to respond quickly in future infections carrying out their function (prevents
● works alongside the innate and specific immune system to with that specific pathogen. viral invasion).
help them destroy pathogens this involves a series of T helper cells also travel to areas of infection and secretes 3. Attach to pathogens by clumping
complement proteins (C1-C9) cytokines that help to recruit monocytes and macrophages to the together to slow the spread of
● COMPLEMENT CASCADE – once the complement system is infected tissue and activate macrophages to cause inflammation pathogen also known as agglutination
triggered, proteins activate each other. and start the process of phagocytosis. 4. Act as Opsonin that is highly specific to
o Various products of the complement cascade have the invading pathogen. By attaching
important functions such as: themselves to that pathogen acting as
▪ Act as opsonin opsonin, they can help the
▪ Trigger inflammation macrophages and neutrophils to
▪ Destroys pathogen recognize and destroy that pathogen.
o The complement system is triggered in 1 or 3 ways
▪ Lectin Pathway – activated directly by CHAPTER 2: SEROLOGY
pathogens ● The scientific study of serum and other body fluids in practice.
▪ Alternative Pathway – activated directly by ● The term usually refers to serologic test.
pathogens ● SEROLOGIC TESTS – blood tests that look for antibodies in the
▪ Classical Pathway – activated by antigen- blood which involves a number of laboratory techniques.
antibody complexes that arise from the o Different types of serologic tests are used to
specific immune system diagnose various disease conditions.
o CYTOTOXIC T CELLS – responsible for killing cells o Serologic tests have one thing in common: they
SPECIFIC IMMUNE SYSTEM that have been infected by pathogens such as focus on proteins made by the immune system.
● Involves the T and B cells, which are lymphocytes that are virally infected cells. To do this, they need to o The process for having the test is the same
free to float around the Lymphatic System and Blood but attach themselves to the infected cells via the T regardless of which technique the laboratory uses
spend most of their time in the lymph nodes and the mucosa cell receptor and HLA class 1 molecule expressing during serologic testing.
associated lymphoid tissue. the relevant antigen protein on the infected cell. ● WHY IS THERE A NEED FOR SEROLOGIC TEST?
● Each pathogens have molecules that are unique to them ▪ Once they are attached to the virally infected o It is helpful to know a little about the immune
known as ANTIGENS cell, they have two killing mechanisms that system and why humans get sick to understand
o T- cells – have T cell receptors that are specific to they can choose form: serologic tests and why they are useful.
a single antigen 1. Granule Exocytosis – spraying the o Antigens are substances that provoke a response
o B-cells – have antibody on their cell membrane infected cell with enzymes that from the immune system. Antigens that
that are also specific to a single type of antigen destroys the membrane and lead to commonly affect people include the following:
● When a new pathogen arrives and causes an infection, the T cell lysis and cell death. ▪ Bacteria
and B cells that are specific to that pathogen needs to be 2. FAS molecule – “self-destruct switch” ▪ Fungi
alerted. that once it’s activated, it causes the ▪ Viruses
o DENDRITIC CELLS – messengers that pick-up cell to undergo apoptosis. ▪ Parasites
antigens at the site of invasion, display them o PLASMA CELLS – B cells that are differentiated to o The immune system defends against antigens by
through the blood of the lymphatic system into become “antibody producing cells”, and their job producing antibodies. These antibodies are
the lymphatic tissue. Once they are there, all the T is to produce antibodies that are specific to the particles that attach to the antigens and
and B cells have a look at the antigen and see invading pathogen. deactivate them, when the doctor tests blood,
whether they recognize it. When T and B cells are ▪ ANTIBODIES – Y-shaped proteins with they can identify the type of antibodies and
found to be specific to an antigen, they become variable region that matches different antigens that are in the blood sample and identify
activated. antigens. The fixed region recognized by the type of infection a patient has.
many cells of the immune system o Sometimes the body mistakes its own healthy
tissue for outside invaders and produces

ESTHER VICTORIA H. TOLENTINO | BSMT 4-3 2

 IMMUNOLOGY AND SEROLOGY SECTION

unnecessary antibodies. This is known as an Key technology ABEI and Nanomagnetic microbead enhances ▪ This test can show your healthcare
autoimmune disorder. Serologic testing can stability and sensitivity of MAGLUMI reagents. provider if your thyroid gland is overactive
detect these antibodies and help the doctor (hyperthyroidism) or underactive
diagnose an autoimmune disorder. (hypothyroidism).
TYPES OF SEROLOGIC TEST ▪ NORMAL VALUES:
✓ T3 = 0.69 – 2.15 ng/mL
1. AGGLUTINATION ASSAY
✓ T4 = 52 – 127 ng/mL
o shows whether antibodies exposed to certain
o TSH, FT3, FT4 – aids in the diagnosis of thyroid
antigens will cause particle clumping.
disorders.
2. PRECIPITATION TEST
▪ FT3 – provides further confirmatory test
o shows whether the antigens are similar by
for monitoring hyperthyroidism to
measuring for the presence of antibody in body
supplement the T3, T4 and thyrotropin
fluids.
assays.
NORMAL RESULTS ABNORMAL RESULTS
▪ FT4 – measures in response to abnormal
The body produces antibodies Antibodies in the blood
TSH test results
in response to antigens. If sample often mean you have
▪ TSH – serves as initial test in thyroid
testing shows no antibodies, it had an immune system
diagnostics
indicates that the body does response to an antigen from PARTS OF MAGLUMI 800 ▪ NORMAL VALUES:
not have an infection. Results either current or past ● Pipettor ✓ FT3 = 2.0 – 4.2 pg/mL
that show there are no exposure to a disease or o Aspirate reagent and sample with high speed ✓ FT4 = 8.9 – 17.2 pg/mL
antibodies in the blood sample foreign protein o Titanium needle for crushproof ✓ TSH = 0.3 – 4.5 uIU/mL
are normal. The presence of certain types o Clot detection and liquid level detection 2. Inflammatory Monitoring:
of antibodies can also mean o Coated with Teflon to prevent carry over o Procalcitonin (PCT) – biomarker associated with
that a patient is immune to o Inner an External washing the inflammatory response to bacterial infection
one or more antigen. ● Automated Cuvette Loader that aid in the risk assessment of critically-ill
o up to 40 modules of 240 tests patients on their first day of ICU admission.
POST-SEROLOGIC TESTING o continuous loading ▪ Guide in antibiotic treatment and monitor
● an antibiotic or another type of medication may help the body ● Sample Area effectiveness of treatment.
to fight the infection. o Up to 40 samples on board with barcode ▪ NORMAL VALUE:
● An additional test may still be required if the doctor thinks o Continuous loading and cooling function ✓ PCT = <0.5 ng/mL
that the patient still has an infection. ● Incubator 3. Tumor Markers:
● The bacteria, virus, parasite, or fungus in the body will o Incubate 13 slots (78 tests at the same time) o Ferritin and Prostate Specific Antigen (PSA)
multiply over time. In response, the immune system will o Temperature is 36.8O C. ▪ Ferritin – used in the evaluation of iron
produce more antibodies. This makes the antibodies easier to ● Reagent Area deficiency and iron overload.
detect as the infection gets worse. o Up to 9 reagents on board with RFID ▪ PSA – aids in the detection of prostate
● Test results may also show the presence of antibodies related o Easy and fast for adding and updating cancer in men aged 50 years or older.
to chronic conditions such as auto-immune disorders. ● Washer ▪ NORMAL VALUES
o Strong magnetic field helps betted separation ✓ Ferritin = 13 – 350 ng/mL
MACHINES o 3 pairs of washing needles with anti-overflow ✓ PSA = <4.0 ng/mL
function 4. Infectious Diseases:
MAGLUMI 800 ● Chamber o Anti-HBs – used to monitor the success of
● New member of Snibe Maglumi series o High sensitivity and low-noise PMT Hepatitis B vaccination
● Fully automated o Cuvettes detection and anti-overflow ▪ Used to monitor convalescence and
● Chemiluminescence Immunoassay Analyzers (CLIA)” TESTS AVAILABLE recovery of Hepatitis B infected individuals.
1. Labelling Technology – determines reaction mode 1. Thyroid Function Tests: ▪ Cut-off Value: 10 mIU/mL
2. Separation Technology – determines sensitivity, o T3 (triiodothyronine) and T4 (thyroxine) – helps ▪ NOTE: Running is performed by MT with
accuracy, and precision of reagents. to control how your body stores and uses energy proficiency training.
ADVANTAGES: (metabolism) OPERATION:
● For small and middle size laboratories or hospitals. ▪ Also helps to control other body processes ● Maglumi 800 can load up to 40 pieces of cuvettes at a time,
● Simple, smart, humanization designed model. such as breathing, heart function, nervous which can run 240 tests. During testing, these can be
● More than enough test menu to meet the different needs system function, body temperature, continuously loaded. Maglumi kit uses RFID technology.
and it is enlarging according to your requirement. cholesterol level, how easily you gain ● Next to the reagent area, there is a sensor which
● Free QC and calibrator helps to reduce the cost per test. weight, brain development, moisture of automatically reads chips on reagent packages and passes
the skin, and menstruation. on the information to the software.

ESTHER VICTORIA H. TOLENTINO | BSMT 4-3 3

 IMMUNOLOGY AND SEROLOGY SECTION

● With SNIBE technology, there are more than 120 kinds of FINECARE FIA METER 11. Press “In” display button.
reagent for choosing. ● a fluorescence immunochromatographic analyzing system 12. Press “Test” display button to start the test.
● Maglumi reagent kits contain a magnetic micro-bead which help diagnose conditions such as infection, diabetes, 13. Wait for 3 minutes for the result.
suspension rotor cardiovascular diseases, renal injury, and cancers. 14. Input corresponding accession number and press “Ok”
● These beads are suspended in solution via the automatic ● the finecare platform provides reliable and quantitative button
mixing that occurs after loading. results of various kinds of analytes in human blood or urine 15. Click “Print” button to print. Record your results.
● In regards to the Maglumi 800, it has capacity for 9 reagents within several minutes.
and can be continuously loaded. KITS
● Samples can be reloaded with different types of sample
tube. SDBIOLINE DENGUE DUO RAPID TEST
● When loading the sample, the barcode reader scans the
barcode on the tube and identifies the sample ID, sending
such information onto the software. The Maglumi 800’s
sample area has a maximum capacity of 40 samples and can
continuously load samples.
● The reagent and sample area are also refrigerated, which
helps increase stability.
● Maglumi software can automatically obtain the worklist via
laboratory information system, or via manually input by
the operator.
PARTS OF FINECARE FIA METER
● A multi-language and user-friendly interface also facilitates ● Designed to detect both dengue virus NS1 Antigen and
● Built-in Thermal Printer
operation. differential IgG IgM antibodies to dengue virus.
o Real-time printing
● After confirmation of the worklist, click “Start” button to ● SD Dengue NS1 Antigen could be useful for early acute phase
● LIS/HIS connection
start the test. samples from day one to five.
● ID Chip Port
● As the test commences, the sample needle pipettes the ● An IgG and IgM antibody test was designed to detect
o Contains all the information of test item and lot
sample and reagents in the cuvette. antibodies appearing in the convalescence phase after day 5
number
● The sample needle is titanium and TEFLON coated which to 14.
● Test Channel
makes it crush proof. ● The main purpose of the test is the simultaneous detection of
o Test cartridge holder
● Internal and external washing pus liquid and clot detection dengue NS1 antigen and IgG IgM together, not separately.
● LCD
all ensure accurate pipetting. ADVANTAGES:
o 3.5 inches touch screen
● After the pipetting is complete, the cuvette is transferred to ● Diagnosis of Dengue NS1 antigen even during window period
the incubator for 10 to 15 minutes TESTS AVAILABLE ● Compensate for the weak point of Antigen single test.
● The incubator has 13 incubation slots available and has ● C-reactive protein CRP – the test measures the level of C- ● Fast result. (15-20 minutes)
precise temperature control to 37OC. reactive protein (CRP) in your blood. CRP is a protein made ● Convenient storage condition. (1-30OC)
● Following incubation, the cuvette is moved into the washing by the liver. It is sent into the bloodstream in response to TEST PROCEDURE
station. Here, the magnetic micro beads are attached to the inflammation
MATERIALS INCLUDED:
magnetic positioned at the back of the cuvette. o Inflammation is the body’s way of protecting
● Test pouch
● Cuvette is then cleaned 3 times with wash buffer to remove tissues if injured or an infection is present.
● Buffer
unconjugated contents. o Cut-off value: <10 ng/mL
● Disposable dropper
● One bottle of concentrated wash buffer is generally diluted OPERATION ● Capillary pipette
to 10L for this purpose. 1. Prepare all materials needed ● Package insert.
● Finally, the cuvette moves into the measuring chamber, 2. Insert ID chip. NOTE: The ID chip contains test item,
which is a darkroom environment. Here, 2 substrates: batch number, standard graph; after finishing test, all *Materials should be prepared before starting the test.
Starter 1 and Starter 2 are added. They react with the information in ID chip was stored in equipment, thus it *All test materials must be used immediately once opened.
present chemiluminescence label: ABEI. is not necessary to insert ID chip of same batch again.
● After measuring, the cuvette is pushed into the waste bag 3. Remove the test device from the foil pouch. 1. Check the expiry date on the backside of the test result
and the test ends. 4. Pipette 5 uL of serum or 8.5 uL of whole blood. NOTE: If the expiry date has passed, use another kit.
SUMMARY OF OPERATION 5. Mix with CRP buffer. 2. Remove the test device from the foil pouch and place it
1. The user loads the desired reagents. 6. Invert for 1 minute. on a flat dry surface.
2. The samples are loaded. 7. Transfer 75 uL of mix buffered and serum into the test 3. Write the corresponding accession number in the test
3. Desired work-list is input manually kit. kit.
4. Press “Start” and wait for results. 8. Click “Standard Test” NOTE: Always check patient’s request and sample if it is
9. Press “Out” display button. correct.
10. Insert cartridge in the test channel

ESTHER VICTORIA H. TOLENTINO | BSMT 4-3 4

 IMMUNOLOGY AND SEROLOGY SECTION

4. For NS1 testing, using a disposable dropper provided, ● Secondary or past dengue infection: IgG POSITIVE *Materials should be prepared before starting the test.
take serum or plasma *All test materials must be used immediately once opened.
5. Add 3 drops of serum or plasma into the sample well
marked “S”. 1. Check the expiry date on the backside of the test
6. At the same time, using the capillary pipette, take pouch.
sample from the collection tube and add drop 10 NOTE: if expiry date has past, use another kit.
microliters of serum or plasma into the sample well for 2. Open the test pouch and set the test device.
antibody test. 3. Write the corresponding accession number on the
7. Put 4 drops of diluent into the round-shaped assay well. test.
8. As the test begins to work, you will see purple color ● Secondary or past dengue infection: IgG/IgM POSITIVE 4. Using a micropipette, take 10 microliters of serum or
move across the result window in the center of the test plasma.
device. 5. Put it into the round sample well.
9. Interpret test results after 15 to 20 minutes. 6. Add 4 drops of buffer into the same round well.
NOTE: DO NOT read the test results AFTER 20 minutes. 7. Read the result in 20 minutes.
Reading too late can give FALSE results. NOTE: DO NOT read the test results AFTER 20 minutes.
TEST RESULT INTERPRETATION Reading too late can give FALSE results.
● One line “C” on result window: NEGATIVE TEST INTERPRETATION
● No “C” line. Repeat test using a new test device: INVALID ● One line “C” result window: NEGATIVE
RESULT

● Indicative of acute stage dengue infection: NS1 Antigen

POSITIVE ● Two color bands (“HIV-1” test line and “C” control line) or
three color bands (“HIV=1” test line, “C” control line, and
NOTE: Each test can be used ONCE. Do NOT try to use the test
faint “HIV-2” test line): HIV 1 POSITIVE
more than once.

SD BIOLINE HIV 1 and 2 RAPID TEST

● Indicative of acute stage dengue infection: NS1 Antigen/IgM

POSITIVE ● Two color bands (“HIV-2” test line and “C” control line) or
three color bands (“HIV-2” test line, “C” control line, and
faint “HIV-1” test line): HIV 2 POSITIVE

● Designed for differential detection of all isotype antibodies

specific to HIV type 1 including subtype O and Type 2
simultaneously.
ADVANTAGES:
● Serum, plasma, or whole blood can be used as specimen.
● Primary Dengue Infection: IgM POSITIVE ● Test result is only 20 minutes
● Storage condition is room temperature for 2 years. ● Perform confirmatory Test: HIV 1 & 2 INFECTION
● Sensitivity is 100% and specificity is 99.8%.
TEST PROCEDURE
MATERIALS INCLUDED:
● Test pouch
● Buffer
● Package insert.
● Capillary pipettes.

ESTHER VICTORIA H. TOLENTINO | BSMT 4-3 5

 IMMUNOLOGY AND SEROLOGY SECTION

● No “C” line on result window. Repeat using a new test kit: 7. Interpret test results at 15-30 minutes.
INVALID RESULT NOTE: If test band is very faint at 15 minutes, read the
results again at 30 minutes.
DO NOT read the test results AFTER 20 minutes. Reading too
late can give FALSE results.

REFERENCES
NOTE: Each test can be used ONCE. Do NOT try to use the test Notes from the discussion of Ms. Christine A. Aguilar, RMT
more than once. Notes from the synchronous session of Ms. Christine A.
Aguilar RMT
SD BIOLINE SALMONELLA TYPHI IgG and IgM FAST EACMC-Cavite Video Presentation for Alternative Medical
● An immunochromatographic assay for the rapid, Technology Internship Program: Immunology and
qualitative, and differential test for the detection of IgG and Serology Section
IgM antibodies to Salmonella typhi in human serum, plasma
of whole blood.
● This test provides only a preliminary test result. Therefore,
more specific alternative diagnosis method must be used in
order to confirm Salmonella typhi infection.
BENEFITS:
● IgM: perfect alternative Widal test
● IgG: examination of past infection
● Test result: 15-30 minutes
● Storage condition: 2-30O C for 24 months.
TEST PROCEDURE
Materials Included:
● Test pouch
● Diluent
● Package insert.
● Disposable test tube
● Loop.

*Materials should be prepared before starting the test.

*All test materials must be used immediately once opened.

1. Check the expiry date on the backside of the test

pouch.
NOTE: if expiry date has past, use another kit.
2. Open the test pouch and set the test device.
NOTE: Allow the kit to come to room temperature prior
to testing.
3. Dispense 4 drops of assay diluent to the disposable
test tube.
4. With a loop provided, dispense 1uL of specimen to the
test tube containing assay diluent.
NOTE: Dip the circular end of the loop into the
specimen, and then carefully place the circular end of
the loop into the test tube. This will add 1uL of
specimen to assay buffer
5. Gently stir the assay buffer with the loop to ensure
adequate mixing of specimen in the assay buffer.
6. Holding the strip vertically, insert the test strip into the
tube containing diluted specimen.

ESTHER VICTORIA H. TOLENTINO | BSMT 4-3 6

 Blood Bank

INTRODUCTION TO BLOOD BANK SECTION blood products to cater the needs of the patients and its
DEFINITION neighboring hospitals within Cavite.
Emilio Aguinaldo College Medical Center – Cavite Blood
Bank is a blood service facility in a hospital, duly licensed SERVICES OFFERED
by the DOH – Center for Health Development whose  ABO/Rh Grouping
u
service capabilities are enumerated below:  Weak D Testing (D Test)
 Advocacy and promotion of voluntary blood  Crossmatching
donation and healthy life;  Direct Antiglobulin Test
 Storage and issuance of whole blood and blood  Resolving ABO/Rh discrepancy
components obtain from a blood center  Investigation of Blood Transfusion Reaction
 Resolving of Incompatible Crossmatch
 Storage and Issuance of Whole Blood and Blood
AFFILIATED BLOOD CENTERS
Components obtained from Affiliated Blood
 Philippine Blood Center (PBC) – as their lead Centers A whole blood with satellite bags attached is centrifuged
blood center
at light spin. Platelet-rich plasma is expressed of the red
 Philippine Red Cross – Cavite Chapter
BLOOD COMPONENTS blood cells into satellite bag. Blood cells are sealed and cut
COMPONENT PREPARATION off. Platelet-rich plasma is centrifuged. Plasma is expressed
BLOOD BANK of the platelets. The platelets are sealed and their tubing is
In modern medical treatments, patients may receive a pint
Emilio Aguinaldo College Medical Center – Cavite Blood cut off. The plasma is either frozen to make fresh frozen
of whole blood or just the specific components of the
Bank is a section in the Department of Laboratory plasma, or frozen and thawed to make cryoprecipitate.
blood that are needed to treat their particular condition.
Medicine where pre-transfusion diagnostic procedures are
This approach to treatment, referred to as blood
conducted with utmost care and accuracy to ensure
component therapy, allows several patients to benefit WHOLE BLOOD
transfusion of safe and quality blood, blood components
from one pint of donated whole blood. Whole blood is the simplest, most common type of blood
or derivatives.
donation. It’s also the most flexible because it can be
The transfusable components that can be derived from transfused in its original form, or used to help multiple
MISSION people when separated into its specific components of red
the donated blood are red cells, platelets, plasma,
Emilio Aguinaldo College Medical Center – Cavite Blood cells, plasma and platelets.
cryoprecipitated, and granulocytes. An additional
Bank Section aims to serve our patients and nearby towns
by provision of safe, high quality blood and blood products component, white cells, is often removed from the
donated blood before transfusion. Whole blood is used to treat patients who need all the
and related services with competent staff, proper components of blood, such as those who have sustained
networking with other blood service facilities and updated significant blood loss due to trauma or surgery, actively
machines/equipment with the support of the bleeding and for exchange transfusion.
management.
VOLUME: 450ml + 63ml anticoagulant
VISION SHELF LIFE: Depends on the anticoagulant used.
Emilio Aguinaldo College Medical Center – Cavite Blood ACD/CPD = 21 days
Bank Section shall be a self-sufficient blood bank facility CPDA-1 = 35 days
that will ensure adequate supply of quality blood and
STORAGE +2°C to +6°C (ref temp.)
TEMPERATURE:

ELAINE JOY A. DELA PEÑA | BSMT 4-3 1

 Blood Bank

RESPONSE TO 1 unit: ↑ 1g/dL of Hgb; Platelets can be prepared from donated whole blood.  Treatment for Von Willebrand’s Disease
TRANSFUSION: ↑ 3% Hct Platelets from several different donors are then combined  Congenital or acquired fibrinogen deficiency
to make one transfusable unit. Alternately, platelets can  FXIII deficiency
PACKED RED BLOOD CELLS be obtained using an apheresis machine which draws
RBCs are usually given because of their hemoglobin blood from the donor’s arm and separates the blood into VOLUME: 10-25 mL
content. PRCBs are used to treat anemia without its components, retains some of the platelets and returns SHELF LIFE/ 1 yr -18°C
substantially increasing the patient’s blood volume. the remainder of the blood to the donor. Using this STORAGE 6 hrs +20°C to +24°C (thawed)
Patients who benefit most from transfusion of red blood process, one donor can contribute about 4-6x as many TEMPERATURE:
cells include those with chronic anemia resulting from platelets as a unit of platelets obtained from a whole blood
kidney failure or gastrointestinal bleeding, and those with donation. RED CELLS, PLATELETS, PLASMA
acute blood loss resulting from trauma. They can also be RED CELLS PLATELETS PLASMA
used to treat blood disorders such as sickle cell disease. Random Donor Single Donor
Storage Refrigerator Agitator Freezer
(Pheresis)
Length Up to 42 5 days 1 year
VOLUME: 250-300 mL VOLUME: 50-70 mL 200-400 mL
days
SHELF LIFE: Open System – 24 hours SHELF LIFE: 5 days
Closed System – same as the STORAGE +20°C to +24°C with constant
LEUKOREDUCTION FILTERS
mother bag TEMPERATURE: agitation (to prevent clumping)
9 9 Leukoreduction filters are expected to reduce the number
STORAGE stored: +2°C to +6°C RESPONSE TO ↑ 5-10 x 10 /L ↑ 30-60 x 10 /L
of leukocytes to less than 5 million cells per unit.
TEMPERATURE: transported: +2°C to +10°C TRANSFUSION:
RESPONSE TO 1 unit: ↑ 1g/dL of Hgb; LEUKOREDUCED BLOOD COMPONENTS
TRANSFUSION: ↑ 3% Hct FRESH FROZEN PLASMA
Plasma is commonly transfused to trauma, burn and shock
patients, as well as people with severe liver disease or
provides coagulation factors for:
 Bleeding
 Abnormal clotting due to massive transfusion
 Patients on warfarin who are bleeding
 Treatment of TTP and HUS
 Factor deficiencies
 ATII deficiency
 DIC

VOLUME: 200-250 mL Post-Storage: used at the bedside; Pre-Storage: used

SHELF LIFE/ 1 yr -18°C shortly after collection of unit
STORAGE 7 yrs -65°C
TEMPERATURE: 6 hrs +2°C to +6°C (thawed) Leukoreduced components are usually given to
SETTLED BLOOD: the plasma is clear without any floating *fresh frozen plasma is thawed before transfusion at +30° -  decrease the incidence of febrile transfusion
red cells +37°C water bath reaction
 decrease the risk of HLA alloimmunization
PLATELET CONCENTRATE CRYOPRECIPITATE  decrease the transmission of CMV
Platelets are most often used during cancer treatment as Cryoprecipitated antihemophilic factor (AHF)
well as surgical procedures such as an organ transplant, in or “Cryo” is the precipitated protein portion that results RED CELL SUSPENSION
order to treat a condition called thrombocytopenia, in after thawing FFP. PRINCIPLE
which there is a shortage of platelets. They are also used A 3-5% red cell suspension is a common reagent in many
to treat platelet function abnormalities. CRYO is used for: serologic procedures. The suspension need not be exactly
 Treatment for Factor VIII deficiency (Hemophilia at 3%; an approximation achieves the appropriate serum-
A) to-cell ration for most test procedures and for an adequate

ELAINE JOY A. DELA PEÑA | BSMT 4-3 2

 Blood Bank

number of red cells so one can read and grade the be either present (+) or absent (-), creating the 8 most
reactions. common blood types (A+, A-, B+, B-, O+, O-, AB+, AB-).

The following steps are intended to help an individual gain FORWARD TYPING
confidence in approximating a 3% red cell suspension Forward typing uses the patient’s red blood cells. All red
visually, both as a suspension of cells and in the blood cells contain antigens that are specific to the
appropriate size of the cell pallet achieved after patient’s blood type. When antibody A (Anti-A) or antibody
centrifugation. B (Anti-B) reagent is added to the patient’s red blood cells,
the antigens on the cells will cause the cells to react with
MATERIALS antibodies.
 Whole blood sample
 Test tubes REVERSE TYPING
 Calibrated plastic pipette Reverse typing uses the patient’s serum. The serum
 Buffered NSS Quality Control Worksheet contains antibodies that react with the reagent red blood
cells which are coated with antigen A (A cells) or antigen B
PROCEDURE PROCEDURE (B cells). The type of antibodies present in the patient’s
1. Transfer 2-3 drops of whole blood or packed red 1. Before performing your daily control testing, serum will determine which reagent red blood cells will
cell to a test tube. inspect all reagents for evidence of cause agglutination, and will therefore confirm the blood
2. Wash red cell 3 times with buffered normal contamination and/or deterioration (i.e marked type.
saline. Completely remove saline. turbidity of reagents; hemolysis of red cells)
3. Transfer 1 drop of washed red cell to a tube and 2. Record the lot number and expiration date of RH TYPING
add 19 drops of buffered normal saline. each reagent and observation on the QC All patients are either Rh D positive or Rh D negative.
4. Mix or cover the tube with parafilm and invert worksheet. Testing for the Rh D factor is done by using reagent Anti-D
gently. 3. Label one test tube of each reagent to be and patient red blood cells. If the patient’s red blood cells
evaluated. contain the D antigen (Rh D factor), there will be
QUALITY CONTROL 4. Add 1 drop of each reagent to the appropriate agglutination when tested with Antibody D (Anti-D) and
PURPOSE tube. the patient is considered Rh D positive. If there is no
To ensure that serologic test reagents are suitably reactive 5. Add 1 drop of freshly prepared known cells to agglutination of cells, then the patient is usually Rh D
each day of use. the appropriate tube. negative.
6. Centrifuge all tubes.
MATERIALS 7. Gently suspend each red cell button and PROCEDURE
examine for agglutination. 1. Label test tubes properly as follows:
 Typing Sera
 Known Cells
8. Record the results on the quality control  -A
worksheet.  -B
 LISS
 AHG  -D
BLOOD TYPING  aC
 Serological Centrifuge
OVERVIEW  bC
 Buffered NSS
Blood typing is a test that determines a person’s blood 2. Make 3-5% red cell suspension from the
 Test tube
type. The test is essential if you need a blood transfusion patient’s sample.
 Quality Control Worksheet
or are planning to donate blood. Not all blood types are 3. Place reagents on the appropriate tubes
compatible, so it’s important to know your blood group.  1 drop of Anti-A to tube “-A”
Receiving blood that’s incompatible with your blood type  1 drop of Anti-B to tube “-B”
could trigger a dangerous immune response.  1 drop of Anti-D to tube “-D”
 2 drops of serum or plasma to tubes
There are four major ABO phenotypes determined by the “aC” and “bC”
presence or absence of two antigens (A and B) on the 4. Add 1 drop of 3-5% red cell suspension to the
surface of red blood cells. In addition to the A and B first 3 tubes.
antigens, there is a protein called the Rh factor, which can

ELAINE JOY A. DELA PEÑA | BSMT 4-3 3

 Blood Bank

5. Add 1 drop of known A and known B to tubes PROCEDURE

“aC” and “bC”. 1. Prepare a 3-5% patient’s red cell suspension.
6. Mix the contents of the tubes gently and 2. Put 1 drop of Anti-D and 1 drop of patient’s RCS.
centrifuge all tubes for 40 seconds. Mix.
7. Gently re-suspend the red cell button and 3. Incubate mixture 15 minutes in a water bath at
examine them for agglutination. +37°C.
4. Centrifuge for 30 seconds at 4500 rpm.
NOTE: 5. Check for agglutination.
To test for infants less than 4 months of age, forward or *If positive for agglutination, report as “Rh
red cell test is permissible. positive”
In the absence of agglutination, proceed to the
next step.
INTERPRETATION 6. Wash mixture 3x with 9% NSS.
1. Agglutination of tested red cells and either 7. Decant the supernatant of last washing. Blot to
hemolysis or agglutination in tests with serum WEAK D TEST (DU TEST) remove excess NSS.
constitute positive test results. OVERVIEW 8. Gently resuspend the packed red cells, then add
2. A smooth cell suspension after resuspension of Weak D – defines all weak expression of RhD, whether 2 drops of AHG reagent.
the cell button is negative test result. quantitative or qualitative (variants). 9. Centrifuge for 30 seconds at 4500 rpm.
3. Any discrepancy between the results in forward 10. Check for agglutination.
and reverse typing should be resolved before the 2 distinct causes of Weak D:
interpretation is recorded for the patient’s ABO 1. Quantitative Weak D – normal antigen is INTERPRETATION
group. expressed but there are less than the number  Rh Positive: presence of agglutination in the
expressed in the normal phenotype. Anti-D tube.
AGGLUTINATION REACTION 2. Qualitative Weak D – usually, a normal number  Rh Negative: absence of agglutination in the
GRADE MACROSCOPICALLY OBSERVED FINDINGS of antigens with genetically abnormal epitopes. Anti-D. This indicates that the cells do not
4+ one solid agglutinate express D and should be classified as D-negative.
3+ several large agglutinates CLINICAL SIGNIFICANCE
2+ medium-sized agglutinates, clear background Anti-D is a clinically significant antibody, and it is important CROSSMATCHING (GEL METHOD)
1+ small agglutinates, turbid background to prevent immunization in females of childbearing OVERVIEW
1w very small agglutinates, turbid background potential to avoid complication of HDFN. Unless there is an urgent need for blood, a crossmatch
w+ or +/- barely visible agglutination, turbid must be performed before a red cell transfusion.
background Patients with partial D red cells are at risk for production
0 no agglutination of anti-D and should receive D-negative blood to be INVITROGEL ABO CROSSMATCH
mf mixed field: mixture of agglutinated and considered candidates of RgIG. INVITROGEL ABO CROSSMATCH demonstrates ABO and D
unagglutinated red cell compatibility testing in enzyme and AHG technique as well
H hemolysis PRINCIPLE as the autocontrol of the recipient all in one gel card.
PH partial hemolysis This reagent will cause agglutination of red cells carrying
the RhD antigen.

MATERIALS
 3-5% red cell suspension
 Anti-D
 Serological centrifuge
 Test tubes
 Calibrated plastic pipette
 AHG
 Worksheet

ELAINE JOY A. DELA PEÑA | BSMT 4-3 4

 Blood Bank

ADVANTAGES OF GEL TECH 8. Incubate the gel card for 15 minutes at 37°C in CROSSMATCHING (MANUAL METHOD)
 Standardized procedure the Gel Card Incubator. OVERVIEW
 Simple and rapid 9. Centrifuge for 10 minutes, read and record Unless there is an urgent need for blood, a crossmatch
 Stable reactions result. must be performed before a red cell transfusion. The
 No washing crossmatch must include immediate spin, incubation at
 Consistent result INTERPRETATION 37°C and the AHG phase.
 Small sample volumes  Positive reactions in microtubes 1, 2, and 3
 Decreased waste demonstrate the presence of A, B, and D IMMEDIATE SPIN
 Ease of interpretation antigens. The IS crossmatch method is designed to detect ABO
 Positive reactions in microtubes 4 and 5 indicate incompatibilities between donor’s red cells and recipient’s
PRINCIPLE incompatibility between the donor and recipient. serum.
As the INVITROGEL ABO CROSSMATCH card containing red  Positive reaction in microtube 6 detects
blood cells is centrifuged under specific conditions, autoantibodies on the recipient’s red cell. It can be used as the sole crossmatch method only if the
agglutinated red blood cells will be trapped in the gel Further tests are required. patient has no present or clinically significant antibodies.
column. The red cells, which are not agglutinated, are  Negative reactions in microtubes 4, 5 and 6
pelleted to the bottom of the microtubes. indicate compatibility. AHG PHASE
The AHG phase is used to detect clinically significant
The reactions are visually read and graded according to AGGLUTINATION REACTION antibodies that might cause transfusion reaction.
their reactivity pattern. GRADE MACROSCOPICALLY OBSERVED FINDINGS
4+ agglutinated red cells form a line at the top of MATERIALS
MATERIALS the gel  Recipient’s sample
 Recipient’s sample 3+ most agglutinated red cells remain in the  Donor’s sample
 Donor’s sample upper half of the gel  LISS
 INVITRO LISS 2+ agglutinated red cells are observed  AHG
 INVITRO Gel Card throughout the length of the gel  Test tube
 Test tube 1+ most agglutinated red cells remain in the  Calibrated plastic pipettor
 Yellow tips lower half of the gel  Serological centrifuge
 Pipettor 0 all the red cell pass through and form a  Dry bath incubator/Water bath
compact button at the bottom of the gel 
 Diamed centrifuge Crossmatching logbook
mf mixed field: agglutinated red cells form a line
 Diamed incubator
at the top of the gel and unagglutinated red
 Crossmatching logbook PROCEDURE
cells form a compact button at the bottom of
I. IMMEDIATE SPIN
the gel microtube
PROCEDURE 1. Label test tube properly.
1. Bring all materials at room temperature before 2. Add 2 drops of patient’s serum/plasma.
testing. 3. Add 1 drop of 3-5% donor’s red cell.
2. Prepare a 0.8% red cell suspension of both the 4. Mix tubes and centrifuge for 40 seconds.
donor’s and recipient’s cells by adding 10uL 5. Read and record the result.
pRBC and 1 mL Invitro LISS.
3. Label the InvitroGel ABO Crossmatch card with INTERPRETATION
patient and donor identification numbers.  Positive result shows agglutination or hemolysis.
4. Remove the aluminum foil cover carefully.  Negative result shows no agglutination or
5. Pipette 50uL of 0.8% recipient’s RCS in hemolysis and indicate a compatible Immediate
microtubes 1, 2, 3 and 6. Spin Crossmatch
6. Pipette 50uL of 0.8% donor’s RCS in microtubes  In case of emergency, above procedure can be
1, 2, 3, 4 and 5. instituted to release a unit of blood.
7. Add 25uL of patient’s serum/plasma in
microtubes 4, 5, and 6.

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 Blood Bank

II. THERMO PHASE unagglutinated red cell  RBCs agglutinate: antihuman antibodies form
1. Add 2 drops of LISS. H hemolysis links between RBCs by binding to the human
2. Mix and incubate for 10-15 minutes. PH partial hemolysis antibodies on the RBCs.
3. Centrifuge for 40 seconds.
4. Read and record the result. MATERIALS
 3-5% red cell suspension
INTERPRETATION  AHG
 Positive result shows agglutination or hemolysis.  Serological centrifuge
 Negative result shows no agglutination or  Test tubes
hemolysis and indicate a compatible Immediate  Calibrated plastic pipette
Spin Crossmatch  Buffered NSS
 In case of emergency, above procedure can be  Worksheet
instituted to release a unit of blood.
PROCEDURE
III. ANTI-HUMAN GLOBULIN PHASE 1. Make a 3-5% red cell suspension.
1. Fill tubes (3/4 full) with 9% NSS and centrifuge 2. Label the tube properly.
for 30 seconds (3x). 3. Dispense 2 drops of AHG to test tube.
2. After the last washing, blot the tube in a gauze 4. Add 1 drop of red cell suspension.
DIRECT ANTIGLOBULIN TEST 5. Centrifuge for 40 seconds.
pad/tissue paper to absorb excess saline.
3. Add 2 drops AHG.
OVERVIEW 6. Examine the cells for agglutination, grade and
The Direct Antiglobulin Test (DAT) is a simple test used to record results.
4. Spin for 30 seconds and read immediately
determine if red cells have been coated in vivo with
macroscopically and microscopically.
immunoglobulin, complement, or both (usually IgG and/or INTERPRETATION
5. Record results.
C3d).  DAT is positive when agglutination is observed
INTERPRETATION either after immediate centrifugation or after
DAT is used primarily for the investigation of hemolytic
 Positive result shows agglutination or hemolysis. the centrifugation that followed room
transfusion reactions, hemolytic disease of the fetus and
 Negative result shows no agglutination or temperature incubation.
newborn (HDFN), autoimmune hemolytic anemia (AIHA),  DAT is negative when no agglutination is
hemolysis and indicates a compatible unit.
and drug-induced immune hemolysis. observed.
INTERPRETATION
PRINCIPLE INDIRECT ANTIGLOBULIN TEST
1. Agglutination of tested red cells and either
hemolysis or agglutination in tests with serum OVERVIEW
constitute positive test results. The Indirect Antiglobulin Test (IAT) demonstrates in vitro
2. A smooth cell suspension after resuspension of reaction between red cells and antibodies. In an IAT,
the cell button is negative test result. serum (or plasma) is incubated with red cells, which are
then washed to remove unbound globulins.
AGGLUTINATION REACTION
GRADE MACROSCOPICALLY OBSERVED FINDINGS PRINCIPLE
4+ one solid agglutinate
3+ several large agglutinates
2+ medium-sized agglutinates, clear background  Blood sample from a patient with immune
1+ small agglutinates, turbid background mediated hemolytic anemia: antibodies are
1w very small agglutinates, turbid background shown attached to the antigens on the RBC
w+ or barely visible agglutination, turbid surface.
+/- background  The patient’s washed RBCs are incubated with
0 no agglutination antihuman antibodies (Coombs reagent).
mf mixed field: mixture of agglutinated and

ELAINE JOY A. DELA PEÑA | BSMT 4-3 6

 Blood Bank

 Recipient’s serum is obtained, containing REFERENCES

antibodies (Ig’s) EACMC-Cavite (Department of Laboratory Medicine), an
 Donor’s blood sample is added to the tube with Alternative Medical Technology Internship Program
serum. Department of Laboratory Medicine (EACMC-Cavite) Video
 Recipient’s Ig’s that target the donor’s red blood Presentation – Blood Bank
cells form antibody-antigen complexes.
 Anti-human Ig’s (Coombs antibodies) are added
to the solution.
 Agglutination of red blood cells occurs, because
human Ig’s are attached to red blood cells.

MATERIALS
 3-5% Group O red cell suspension
 Patient’s serum (or plasma)
 AHG
 Serological centrifuge
 Test tubes
 Calibrated plastic pipette
 Buffered NSS
 Incubator
 Worksheet

PROCEDURE
1. Add 2 drops of serum (or plasma) to a properly
labeled tube.
2. Add 1 drop of 3-5% Group O red cell suspension.
3. Centrifuge and observe for hemolysis and
agglutination. Grade and record.
4. Incubate at 37°C for 30 to 60 minutes.
5. Centrifuge and observe for hemolysis and
agglutination. Grade and record.
6. Wash the red cells 3-4 times with saline and
completely decant the final wash.
7. Add 2 drops of AHG to the dry cell button.
8. Centrifuge and observe for hemolysis and
agglutination. Grade and record.

INTERPRETATION
 POSITIVE – presence of agglutination and/or
hemolysis after incubation at +37°C and at AHG
phase
 NEGATIVE – absence of agglutination and/or
hemolysis after incubation at +37°C and at AHG
phase

ELAINE JOY A. DELA PEÑA | BSMT 4-3 7

 CLINICAL CHEMISTRY SECTION

CLINICAL CHEMISTRY ● Glucose, BUN, Creatinine, Uric Acid, ALT, AST, Lipid GLUCOSE, BUN, CREATININE, URIC ACID:
● Refers to the biochemical analysis of body fluids Profile (Total Cholesterol, Triglyceride, HDL, LDL) ● Limitations: If the value exceeds the linearity, the
● Uses chemical reactions to determine the levels of specimen should be diluted 2-fold with distilled
various chemical compounds in body fluids GLUCOSE water, assay repeated, and results multiplied by the
● Most common specimens: Blood and Urine ● Hyperglycemia and Hypoglycemia DF of 2.
● Methodology: Glucose oxidase
EACMED CLINICAL CHEMISTRY SECTION ● Turnaround Time: 2 hours ALANINE AMINOTRANSFERASE (ALT)
1. ROUTINE BLOOD CHEMISTRY ● Normal range: 3.89 – 5. 83 mmol/L ● Increased: hepatitis, cirrhosis, and obstructive
● Fasting/Random Blood Sugar, BUN, Creatinine, Uric ● Linearity: 16.6 mmol/L jaundice
Acid, ALT, AST, Lipid Profile (Total Cholesterol, ● Method: Enzymatic
Triglyceride, HDL, LDL) BLOOD UREA NITROGEN (BUN) ● Turnaround Time: 2 hours
2. SPECIAL BLOOD CHEMISTRY ● Increased: acute and chronic nephritis, intestinal and ● Normal range: 3 – 35 U/L
urinary obstruction, uremia ● Linearity: 600 U/L
● Total Protein, Albumin, Alkaline Phosphatase, Lipase,
Amylase, Total/Direct/Indirect Bilirubin, Lactate ● Decreased: acute liver destruction, pregnancy
Dehydrogenase ● Methodology: Modified urease ASPARTATE AMINOTRANSFERASE (AST)
3. ELECTROLYTES ● Turnaround Time: 2 hours ● In myocardial infarction, value may begin to rise within
● Normal range: 8 – 23 mmol/L 6-8 hours after onset, peak within 2 days, and return
● Sodium, Potassium, Chloride, Calcium, Phosphorus,
● Linearity: 49.99 mmol/L to normal by the 4th/5th day of post-infarction.
Magnesium
● Increased: hepatitis, liver necrosis, cirrhosis, and liver
4. OTHERS metastasis
CREATININE
● Glycosylated Hemoglobin (HbA1c) ● Method: Enzymatic
● Increased: renal disorders such as glomerulonephritis,
● Cardiac Markers (CKMB, Troponin-I, Myoglobin) ● Turnaround Time: 2 hours
pyelonephritis, acute tubular necrosis, and urinary
obstruction ● Normal range: 3 – 35 U/L
ANALYZERS ● Methodology: Enzymatic ● Linearity: 600 U/L
1. Cobas c111 Chemistry Analyzer
● Turnaround Time: 2 hours
2. Altair 240 Chemistry Analyzer ALT & AST
● Normal range:
3. Easylyte Plus Na/K/Cl Analyzer  Male: 79.56 – 114.92 umol/L ● Limitations: Dilute 1-part sample with 9 parts
4. Easylyte Expand Na/K/Cl/Ca Analyzer distilled water and re-assay. Multiply the result by
 Female: 53.04 – 97.24 umol/L
5. Finecare FIA Meter 10.
● Linearity: 2652.6 umol/L
6. Exdia TRF Cardiac Analyzer
URIC ACID TOTAL CHOLESTEROL
ALTAIR 240 ● Increased: at higher risk of heart disease or stroke
● Random-access analyzer ● Increased: gout, kidney disease, and cancer
● Methodology: Uricase ● Method: Modified Trinder
● Throughput: up to 400 ● Turnaround Time: 2 hours
tests per hour ● Turnaround Time: 2 hours
● Normal range: ● Normal range: 3.64 – 5.2 mmol/L
● Colorimetric and ● Linearity: 19.43 mmol/L
Turbidimetric  Male: 202.23 – 416.36 umol/L
measurement  Female: 142.75 – 339.04 umol/L
● Linearity: 1189.6 umol/L

PREPARED BY: RICCI ANNE V. LAUREZO | BSMT 4-3 1

 CLINICAL CHEMISTRY SECTION

TRIGLYCERIDE 3. Recap the vials. 20. Click “Search”

● Diagnosis of primary and secondary 4. Gently swirl the vials. 21. Select Test then “Yes”
hyperlipoproteinemia 5. Allow the controls to stand at room temperature (18- 22. Log results.
● Method: Glycerylphosphate oxidase (GPO) 250C) for at least 15 minutes.
● Turnaround Time: 2 hours
● Normal range: 0.33 – 1.65 mmol/L
● Linearity: 11.3 mmol/L

HDL CHOLESTEROL
● Method: Homogenous method for direct HDL
measurement
● Turnaround Time: 2 hours 6. Place 200 uL of each control level in the sample cups. 23. The rest of the aliquots are sealed with a parafilm
● Normal range: and stored ar -25 to -150Cup to 28 days.
 Male: 0.78 – 1.82 mmol/L 24. Frozen aliquots of QC samples are thawed at room
 Female: 0.78 – 2.21 mmol/L temperature for 30 minutes prior to testing.
● Linearity: 0.03 – 4.14 mmol/L
RUNNING OF SAMPLES
LDL CHOLESTEROL Table 2. Running of samples
● Computed: Friedewald equation

EQUATION: 7. Click worklist on the main screen.

LDL–C = Total Cholesterol – (TAG/2.2) – HDL–C 8. Click “QC”.
9. Select the test(s).
RUNNING OF CONTROLS 10. Click “Save WL”.
PREPARATION OF QC SAMPLES
Table 1. Preparation of QC samples 1. Verify the sample and the request.
2. Label the sample cup with the sample ID and name
of the patient.

11. Place the controls onto the sample tray.

1. Stand the control sera from the storage refrigerator

at room temperature for 10 minutes prior to
reconstitution.
3. Add at least 200 uL of patient’s serum into the
sample cup.

12. Click “Next” twice

13. Click “Start Random”
14. Click “Results”
15. Click “QC/Std Results”
16. Click “Store”
2. Add the required volume of distilled water (5 mL) to 17. Click “Yes” then “Ok”
the control sera using a volumetric pipette. Then, recap 18. Select “Memory Files” 4. Define the orders.
and gently swirl the vials. 19. Click “Std/QC Archive” 5. Click “Worklist” on the main screen.

PREPARED BY: RICCI ANNE V. LAUREZO | BSMT 4-3 2

 CLINICAL CHEMISTRY SECTION

6. Enter the patient’s name. ● Decreased: loss of blood, sprue, nephrotic syndrome, ● Turnaround Time: 3 hours
7. Select the gender of the patient. severe burns, salt retention syndrome, and ● Normal range: 28 – 100 U/L
8. Select the test(s). Kwashiorkor ● Linearity: 3 – 1500 U/L
9. Click “Save in WL”. ● Increased: severe dehydration and multiple myeloma
● Methodology: Biuret LIPASE
● Turnaround Time: 3 hours ● After acute pancreatitis, the lipase activity increases
● Normal Range: 66 – 87 g/L within 4-8 hours, reaches a peak after 24 hours, and
● Linearity: 2.0 – 120 g/L decreases after 8-14 days.
● Methodology: Enzymatic Colorimetric
ALBUMIN ● Turnaround Time: 3 hours
● Decreased: reduced CHON intake, severe ● Normal range: 13 – 60 U/L
burns/inflammation, malabsorption of amino acids, ● Linearity: 3 – 300 U/L
10. Load sample.
nephrotic syndrome, protein loss via stool
11. Close the cover.
● Increased: dehydration or severe diarrhea LACTATE DEHYDROGENASE
● Methodology: Bromcresol Green ● Highest levels: megaloblastic anemia, disseminated
● Turnaround Time: 3 hours carcinoma, and shock
● Normal Range: 39.7 – 49.9 g/L ● Moderately increased: muscular disorders, nephrotic
● Linearity: 2.0 – 60 g/L syndrome, and cirrhosis
● Mild increase: myocardial and pulmonary infarction,
GLOBULIN leukemia, hemolytic anemia, and non-viral hepatitis
● Computed ● Methodology: UV Assay
12. Click “Next” twice. EQUATION: ● Turnaround Time: 3 hours
13. Click “Start Random”. Globulin = Total Protein - Albumin ● Normal range:
14. To retrieve results, click “Results” on the main  Females: 135 – 214 U/L
screen. A/G RATIO  Male: 135 – 225 U/L
15. Search for the patient’s name.  Children (2-15 years): 120 – 300 U/L
● Computed
16. Validate the result.  Newborns (4-20 days): 225 – 600 U/L
EQUATION:
17. Perform delta check. ● Linearity: 10 – 950 U/L
A/G Ratio = Albumin/Globulin
18. Print the result at the back or the worksheet.
TOTAL BILIRUBIN
ALKALINE PHOSPHATASE (ALP)
ANALYZERS ● Increased: anemia, cirrhosis, reaction to a blood
COBAS C111 ● Increased: obstructive jaundice, Paget’s disease,
transfusion, Gilbert syndrome, Viral hepatitis, alcoholic
hyperparathyroidism, rickets, osteomalacia, fractures,
● Continuous random- liver disease, gallstones
malignant tumors
access analyzer ● Methodology: Colorimetric Diazo
● Methodology: Colorimetric
● Throughput: up to ● Turnaround Time: 3 hours
● Turnaround Time: 3 hours
100 tests per hour ● Normal range:
● Normal range:
● Absorbance and  Newborn:
 Male: 40 – 129 U/L
Photometer and ▪ 24 hours: ≥ 137 µmol/L
 Female: 35 – 104 U/L
optionally an ISE ▪ 48 hours: ≥ 222 µmol/L
● Linearity: 3 – 1200 U/L
module that uses ion selective potentiometry. ▪ 84 hours: ≥ 290 µmol/L
● Total Protein, Albumin, Alkaline Phosphatase, Lipase,  Adult: ≤ 21 µmol/L
Amylase, Total/Direct/Indirect Bilirubin, Lactate AMYLASE  Children with age ≥ 1 month: ≤ 17 µmol/L
dehydrogenase, Calcium, Magnesium, Phosphorus. ● Acute pancreatitis, inflammatory phase of chronic ● Linearity: 2.5 – 650 µmol/L
pancreatitis, renal failure, tumor of the lungs or
TOTAL PROTEIN ovaries, pulmonary inflammation, diseases of the DIRECT BILIRUBIN
salivary gland, diabetic ketoacidosis, cerebral trauma,
● Increased: alcohol, infectious hepatitis, drug reactions,
macroamylasemia
and autoimmune disorders
● Methodology: Colorimetric

PREPARED BY: RICCI ANNE V. LAUREZO | BSMT 4-3 3

 CLINICAL CHEMISTRY SECTION

● Methodology: Colorimetric Diazo ● Hyperphosphatemia: Hypoparathyroidism, Vitamin D 2. Place at least 200 uL of patient’s serum in the sample
● Turnaround Time: 3 hours intoxication, and renal failure with decreased cup.
● Normal Range: ≤ 5 µmol/L glomerular phosphate filtration
● Hypophosphatemia: rickets, hyperparathyroidism,
MAGNESIUM and Fanconi’s syndrome.
● Increased: renal failure, acute diabetic acidosis, ● Methodology: Molybdate UV
dehydration, Addison’s disease ● Turnaround Time: 3 hours
● Decreased: chronic alcoholism, glomerulonephritis, ● Normal range: 0.81 – 1.45 mmol/L
malabsorption, acute pancreatitis, tubular ● Linearity: 0.10 – 6.46 mmol/L 3. Click “Order” on the main screen.
reabsorption defects 4. Enter the patient’s name.
● Methodology: Colorimetric with Chlorophosphonazo RUNNING OF CONTROLS 5. Select the test(s).
III Table 3. Running of controls 6. Click “✓”.
● Turnaround Time: 3 hours 7. Place the sample onto the sample tray.
● Normal range: 8. Press the start button.
 Newborn: 0.62 – 0.91 mmol/L 9. Write the results on the worksheet.
 5 months – 6 years: 0.70 – 0.95 mmol/L
 6 – 12 years: 0.70 – 0.86 mmol/L ANALYZERS
 12 – 20 years: 0.70 – 0.91 mmol/L EASYLYTE PLUS/EASYLYTE EXPAND
 Adults: 0.66 – 1.07 mmol/L ● Automated, microprocessor
 60 – 90 years: 0.66 – 0.99 mmol/L -controlled analyzer
 >90 years: 0.70 – 0.95 mmol/L ● Ion Selective Electrode
● Linearity: 0.15 – 2.5 µmol/L ● Serum and Urine:
 Na, K, Cl
CALCIUM ● Serum:
● Increase: in serum PTH or vitamin D are associated  Ionized Ca, pH
with hypercalcemia 1. On the main screen, click “Workplace”. ● Analysis takes 55-60 seconds
● Hypercalcemia: multiple myeloma and other 2. Click “QC Status”. ● Serum: 100 µL
neoplastic diseases 3. Click “+”. ● Urine: 400 µL
● Hypocalcemia: hypoparathyroidism, nephrosis, and 4. Click the test analyte then select the control level.
pancreatitis 5. Click “>>” to proceed. EASYLYTE PLUS
● Methodology: 5-nitro-5’-methyl-BAPTA (NM-BAPTA) 6. Place the QC samples on the instrument ● Na, K, Cl
● Turnaround Time: 3 hours consecutively.
● Normal range: 7. Press the start button. EASYLYTE EXPAND
 Children (0 – 10 days): 1.90 – 2.60 mmol/L 8. Log the QC results on the designated log sheet. ● Na, K, Cl, Ionized Ca
 Children (10 days – 2 years): 2.25 – 2.75 mmol/L
 Children (2 – 12 years): 2.20 – 2.70 mmol/L RUNNING OF SAMPLES
 Children (12 – 18 years): 2.10 – 2.55 mmol/L RUNNING OF CONTROLS
Table 4. Running of samples Table 5. Running of controls
 Adults (18 – 60 years): 2.15 – 2.50 mmol/L
 Adults (60 – 90 years): 2.20 – 2.55 mmol/L
 Adults (>90 years): 2.05 – 2.40 mmol/L
● Linearity: 0.20 – 5.0 mmol/L

PHOSPHORUS
NOTE! Increase in Phosphorus level causes a decrease
in calcium level.
1. When “Analyze Blood?” appears on the daily screen,
1. Label the sample cup with the sample ID. press the No Button thrice until “Second Menu?”
appears.

PREPARED BY: RICCI ANNE V. LAUREZO | BSMT 4-3 4

 CLINICAL CHEMISTRY SECTION

2. Press the Yes button and choose “Quality Control?”. ● Linearity: 2.0 – 300.0 ng/mL ANALYZERS
3. Press “Yes” to run the control. FINECARE FIA METER
3. Run the Normal control. (The sample probe goes MYOGLOBIN ● A portable equipment for
down) ● Turnaround Time: 2 hours fluorescence detection to
4. Gently swirl the control. ● Normal value: ≤ 80.0 ng/mL quantify concentration of various
5. Probe in control. Press the Yes button. Make sure the ● Linearity: 10.0 – 500.0 ng/mL blood analytes.
probe hole is below the sample through out the ● HbA1c, CKMB, Troponin-I
aspiration. ANALYZER SYSTEM CHECK
6. Validate the results. GLYCOSYLATED HEMOGLOBIN (HbA1c)
Table 7. Exdia TRF Plus System Check
7. Write the QC result in the designated log sheet.
● Recommended for patients with diabetes every 2-3
months as part of the patient management program.
RUNNING OF SAMPLES ● Turnaround Time: 2 hours
Table 6. Running of samples ● Reference Rage: 4.0 – 6.5%
● Linearity: 4.0 – 14.5%

ANALYZER SYSTEM CHECK

Table 9. Finecare FIA Meter System Check
1. On the main screen, press “Quality Control”.
2. Click “Quality System Check”.
3. Push open the cassette holder. Insert the QC kit.
1. Verify the sample and patient’s request. 4. Click “Run” on the screen.
2. When “Analyze Blood?” is displayed on the screen, 5. The result will appear on the screen. Click print.
press Yes. 6. Remove the cassette from the machine.
3. Enter the Patient ID. Press Yes. 7. Record the results in the designated log sheet.
4. Probe in blood. Make sure the probe hole is below
the sample through out the sampling operation. RUNNING OF SAMPLES 1. Click “Quality Testing” on the screen.
5. Validate the results. Attach the print out to the Table 8. Running of samples 2. Insert the QC cartridge into the cassette holder.
worksheet. 3. Click “Quality” on the screen.
4. Click “Print”.
ANALYZERS 5. Record the result in the designated log sheet.
EXDIA TRF PLUS
● Uses Time–Resolved Fluorescence RUNNING OF SAMPLES
based on Europium as conjugate, Table 10. Running of samples
minimizing background
fuorescence.
● Acute Myocardial Infarction and 1. Open the foil pouch. Use the kit immediately after
cardiac muscle damage. opening.
 Troponin-I, CKMB, Myoglobin 2. Draw 80 μl of the sample and load it onto the sample
well of the test cassette.
3. On the machine’s screen, click “New Test”.
TROPONIN – I
4. Enter the patient’s name.
● Turnaround Time: 2 hours 5. Click “Enter”. 1. Gently invert the sample.
● Normal value: <0.06 ng/mL 6. Push open the cassette holder. Insert the test 2. Draw the required volume of blood. (10 μl of whole
● Linearity: 0.03 – 30.0 ng/mL cassette. blood for HbA1c)
7. Close the cassette holder. Insert the test cassette. 3. Add the sample to the detection buffer.
CKMB 8. Click “Run” on the screen. 4. Mix the sample for 1 minute by tapping or inverting
● Turnaround Time: 2 hours 9. Click “Print” after 15 minutes later when the result the tube.
● Normal value: ≤ 5.0 ng/mL will show on the display screen.

PREPARED BY: RICCI ANNE V. LAUREZO | BSMT 4-3 5

 CLINICAL CHEMISTRY SECTION

5. Take 75 μl of the sample mixture and load it onto the management of privately-owned drug treatment SCREENING DRUG TESTING LABORATORY
sample well of the test cartridge. rehabilitation centers and drug testing networks and ORGANIZATION
6. Click “Standard Test” on the machine’s screen. laboratories throughout the country, in coordination
7. Click “Out” to release the cartridge holder. with DSWD and other agencies.
8. Insert the test cartridge into the cartridge holder.
9. Click “Test”. ART. III SEC. 36: AUTHORIZED DRUG TESTING
10. Enter the patient’s name. ● Drug testing shall be done by any government forensic
11. Print the result. laboratory or lab accredited and monitored by DOH.
● DOH shall set the price/cost of drug testing
NEQAS  Government – not more that PHP 250.00
● National External Quality Assessment Scheme  Private – not more than PHP 300.00
● Reference Laboratory: Lung Center of the Philippines ▪ Board Regulation No. 9, Series of 2007
● 12 samples (1 sample tested every month) for glucose, ● Shall use two methods of testing:
cholesterol, creatinine, triglyceride, BUN, BUA,  Screening Test
Albumin, Na, K, and Cl  Confirmatory Test
● NEQAS results are submitted online HEAD OF THE LABORATORY
SEC. 39 OF R.A. 9165 ● A person who is accredited to supervise a drug testing
● The DOH shall be tasked to license and accredit drug laboratory.
testing centers in each province and city. ● A certified Clinical Pathologist
● There shall be a control regulations, licensing and ● Licensed Physician with certification in Clinical
accreditation division under the supervision of the Laboratory Management Training conducted by DOH
DOH for this purpose. ● Maximum number of SDTL a physician can handle is 10
(physically feasible to supervise; within 5km radius)
DRUG TEST KITS VALIDATION
● DOH is tasked to validate and register drug test kits ANALYST
RECONSTITUTION OF NEQAS SAMPLES thru Center For Device Regulation, Radiation Health
1. Stand the sample from the refrigerator at room ● Responsible for verifying the chain of custody, perform
And Research (CDRRHR) of the Food and Drug
examination, certify results, and implement quality
temperature for 10 minutes prior to reconstitution. Administration (FDA).
assurance program.
2. Reconstitute the sample that is due for the month on the ● The NRL, East Avenue Medical Center perform
● Either a full-time licensed chemist, medical
day that you will run it. technical evaluation of drug test kits.
technologist, pharmacist, or chemical engineer with
3. Open the vial very carefully avoiding any loss of material. appropriate training in screening test procedures for
Using a volumetric pipette, reconstitute each vial with 5 mL MANDATORY DRUG TESTING
dangerous drugs.
of distilled water. 1. Applicants for firearm’s license
 DOH shall recognize the training program.
4. Replace the stopper and allow the sample to stand at 18- 2. Officers and members of the military, police and other
250C for at least 20 minutes, swirling occasionally. law enforcement agencies (annual drug testing)
AUTHORIZED SPECIMEN COLLECTOR
5. Before sampling gently swirl the sample for several times 3. Persons charged before the prosecutor’s office with a
● Instructs, assists Client/Donor/Subjects at a collection
to ensure homogeneity. criminal offense having an imposable penalty of
site.
imprisonment of not less than 6 years and 1 day.
● Handles, transports, and stores specimen correctly
MANUAL OF OPERATIONS FOR DRUG TESTING 4. Candidates for public office
● Ensure specimen integrity and security of the
LABORATORIES 5. Person apprehended or arrested for violation of R.A. procedure
9165. ● Initiates documentary entries in CCF
REPUBLIC ACT 9165
● Otherwise known as the “Comprehensive Dangerous ● Performs initial specimen validity
Drugs Act of 2002” mandates the DOH to oversee and RANDOM DRUG TESTING
monitor the integration, coordination and supervision ● Secondary and Tertiary Students SPECIMEN COLLECTION
of all drug rehabilitation, intervention, after-care and ● Officers and employees of government and private ● Considered as the “weakest link” of DT Program.
follow-up programs, projects and activities as well as officers ● Proper specimen collection is essential for legally and
the establishment, operation, maintenance and scientifically defensible drug test result; failure to

PREPARED BY: RICCI ANNE V. LAUREZO | BSMT 4-3 6

 CLINICAL CHEMISTRY SECTION

follow any of these steps will invalidate the results and *WOD= Window of Detection Department of Laboratory Medicine: An Alternative
make them indefensible in legal settings. Medical Technology Internship Program Video
● Sample collected: Urine ANALYTICAL METHODS Presentation – Clinical Chemistry Section
● Types: 1. SCREENING
 Single Specimen Collection – single 60 mL bottle ● The methods used may be:
 Split Specimen Collection – 2 separate bottles;  Registered testing kits approved by FDA-DOH
30 mL each using immunoassay technique
 Instrumented Screening method
METHODS OF SPECIMEN COLLECTION ▪ Immunoassay: ELISA, FPIA
OBSERVED COLLECTION ▪ Chromatography: TLC, HPLC
● In the presence of ASC
2. CONFIRMATORY
● GC-MS (Gas Chromatography-Mass Spectrometry) –
UNOBSERVED COLLECTION
gold standard
● In the absence of ASC ● LC-MS – Platinum reference standard
● Submitted samples
● Subject to validity tests IDTOMIS (INTERGRATED DRUG TEST OPERATIONS
MANAGEMENT INFORMATION SYSTEM)
CONDITIONS WHEN UNOBSERVED SPECIMEN ● ICT infrastructure established by government to
COLLECTION IS ALLOWED provide a standard and effective system for conducting
● When the client/donor is: drug tests and ensuring quality in the operation of
 Physically unable to go to the laboratory or DTL’s.
designated collection site ● A software for DTL’s to use and connect to DOH.
 Involve in crime scene ● IDTOMIS automates the processes from accreditation,
 Involve in post-accident drug test and rehab operations, quality assurance and
 Critically ill monitoring.

DRUGS TESTED IN SCREENING DRUG TESTING INTERPRETATION OF RESULTS

LABORATORY
1. Methamphetamine (Shabu)
2. Tetrahydrocannabinol (Marijuana)

DRUG OF ABUSE CUT-OFFS AND WINDOWS OF

DETECTION (URINE)
Table 11. Drugs of abuse cut-offs and windows of
detection
DRUGS WOD INITIAL CONFIRMATION
SCREEN (ng/mL)
(ng/mL)
Marijuana 2 days – 50 15
3 weeks
PRINCIPLE OF TEST
Cocaine 2-3 300 100
days
● Competitive Binding Immunoassay
Opiates 2-3 2000 2000
days
Amphetamine 2-3 1000 250 REFERENCES
days Notes from the discussion of Mr. Anthony B. Dirain III, RMT
Methamphetamine 2-3 500 250 Notes from the discussion of Ms. Christine A. Aguilar, RMT
days
Emilio Aguinaldo College Medical Center (EACMC) – Cavite
PREPARED BY: RICCI ANNE V. LAUREZO | BSMT 4-3 7
 MICROBIOLOGY SECTION (PART 1)
CULTURE MEDIA PREPARATION • Ex: Thioglycollate broth, Selenite F broth
TYPES OF MEDIA: 6. TRANSPORT MEDIA
ACCORDING TO CONSISTENCY: • For temporary storage of specimens being
transported to the laboratory for cultivation
1. LIQUID (BROTH) MEDIA
• Ex: Cary Blair Transport Medium, Amies Transport
• Propagation of large no. of organisms,
Medium
fermentation studies and various other tests Weigh an amount of the powder according to the
• Ex: BHI broth, thioglycollate manufacturer's instruction and place in an Erlenmeyer
2. SEMI-SOLID MEDIA SAFETY PRECAUTIONS flask
• Cultivation of microaerophilic bacteria or for ✓ Use of PPE is required during the conduct of preparing Step 2 Step 3
determination of bacteria motility media.
3. SOLID MEDIA ✓ Dehydrated culture media are highly hygroscopic.
Keep container tightly closed once opened
• Allows media to grow in physically informative or
✓ Dehydrated culture media are for lab use only
useful ways
✓ Wash hands after every preparation
• Ex: MHA, NA, BAP
✓ Do not eat and use for food
✓ Observe aseptic techniques Label the flask with the Add water
ACCORDING TO FUNCTION ✓ Do not use culture media after expiry date name of the medium and
1. GENERAL (BASAL/BASIC) MEDIA ✓ Check the instruction label before storing the culture volume
• Supports the growth of a wide variety of media. Step 4 Step 5
microorganism types and lack inhibitory property (putol yung vid)
• Ex: NA, TSA nutrient agar, tryptic soy agar BASIC GUIDELINES
2. SELECTIVE MEDIA STORAGE
• Favors the recovery of specific types or genera of
• Keep container tightly closed once opened
microorganisms and inhibits other members of a
mixed microflora thiosulfate citrate bile salt sucrose, • Do not use culture media after expiry date
gentamycin blood agar, bacillus cereus agar • Check the instruction label before storing the culture Cover with aluminum foil
• Ex: MAC, TCBS, GBA, BCA and put a tape on flask
3. DIFFERENTIAL/INDICATOR MEDIA media
Step 6 Step 7
• Possess certain ingredients that enable
presumptive identification of a specific genus or STERILIZATION
species either from a pure or mixed culture • Sterilization by heat is the most used
• Ex: MAC, TCBS • Media containing carbohydrates should not be
4. ENRICHED MEDIA autoclaved at temperature exceeding 116ºC to 118 ºC
• Supports the growth of a wide variety of • In some liquid media, warming may lead to loss of
microorganism including some of the more activity of some compound. In these cases, sterilization Autoclave at 121ºC for 15 After autoclaving, cool to
fastidious ones by filtration must be performed minutes 45ºC to 50ºC
• BAP, CAP Step 8
5. ENRICHMENT MEDIA MEDIA PREPARATION
• Used to increase the relative concentration of Step 1
certain microorganisms, including some of the
more fastidious ones

GAIL YVANA D. SARMIENTO | BSMT 4-3 1

 Microbiology Section (Part 1)

coli ATCC 25922 and Pseudomonas aeruginosa

ATCC 27853 or organism isolated from clinical
specimens that were previously well identified.
4. Record quality control results as to whether the
strain used produces the appropriate biochemical
reactions/color on the test medium
For BAP, aseptically add 5- Measure the blood in a
10% sterile defibrinated sterile test tube or use a
sheep blood. Allow the sterile serological pipette PROPER COLLECTION AND PROCESSING OF
blood to equilibrate at or a graduated cylinder SPECIMENS
room temperature before How to successfully recover bacteria from clinical
adding specimens?
Dispense/pour about 20 mL into sterile petri dishes.
1. Advance planning
Depth of agar layer must be 4 mm
2. Collect appropriate and adequate amount of
Step 11 Step 12
specimen.
3. Proper packaging and transport
4. Ability of the laboratory to accurately perform the
diagnostic test.

GENERAL GUIDELINES
Once solidified, label the Store the plates at 4ºC in 1. Follow universal precaution guidelines
plates with the name of an inverted position or for 2. Treat all specimens as potential biohazards
the media and preparation longer shelf life, place in a 3. Collect specimen before antibiotic treatment
Step 9 date sterile tight plastic bag (for 4. Collect from appropriate site
up to 2-3 months) 5. Practice proper and aseptic collection technique
6. Ensure sufficient quantity
QUALITY ASSURANCE 7. Follow recommended time of transport
I. STERILITY TEST 8. Label specimen accordingly
1. Incubate two tubes or plates from each 9. Accompany specimen with complete request form
Mix by gentle swirling to autoclaved or filter-sterilized medium overnight
avoid formation of at 36±1ºC CLINICAL SPECIMENS FOR AEROBIC CULTURE
bubbles 2. Check tube or plate for growth after 24 hours of 1. STERILE SAMPLES
Step 10 incubation a. Blood
3. Record quality control results in the designated b. CSF
log sheets c. Effusions
II. CULTURE RESPONSE TEST/GROWTH DEPENDENCE 2. NONSTERILE SAMPLES
a. Upper respiratory tract specimens
TEST
b. Lower respiratory tract specimens
1. For plated media: c. Urine
1.1. Inoculate at least one strain to test for ability d. Exudates
of a media to support growth of the target
pathogen
BLOOD
2. For biochemical media:
2.1. Inoculate at least one organism that will When is a blood culture requested?
produce a positive reaction and at least one 1. Acute illnesses
organism that will produce a negative 2. Fever of unknown origin
reaction 3. Acute infective endocarditis
3. Use quality assurance organisms such as 4. Suspected bacterial endocarditis
Staphylococcus aureus ATCC 25923, Escherichia

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 2

 Microbiology Section (Part 1)

Components of a blood culture broth 1. Abscess

1. 1% gelatin • Collection method:
• Enhance growth of N. meningitidis o General: remove surface exudate by wiping
2. 0.1% Bacto agar with sterile saline or 70% alcohol
• Enhance growth of anaerobic organism o Open wound: Aspirate or swab deep into the
3. 0.025% SPS lesion to firmly sample the lesion’s “fresh
• Anticoagulant border”
2. Throat swab o Close wound: Aspirate
• Inhibits activity of complement and lysozyme
• Detects phagocytosis 2. Ear/eye discharge
3. Tissue
• Inactivates therapeutic concentration of
aminoglycosides • Tissue collection is an invasive procedure and
requires surgery by a trained physician
• Place the specimen in a sterile container on sterile
COLLECTION METHOD
gauze moistened with sterile saline
1. Find puncture site. Clean site using 70% alcohol,
• Transport to the laboratory. DO NOT
followed by an iodine solution.
REFRIGERATE.
2. Sterilize the rubber stopper using alcohol swab
• Tissue submitted in formalin is unacceptable for
3. Collect the required amount of blood LOWER RESPIRATORY TRACT
culture
4. Inoculate collected blood into the blood culture 1. Sputum
broth 2. Endotracheal aspirate General rule on collection: clean site first and disinfect
5. Label the specimen property 3. Bronchoalveolar lavage surrounding before collecting sample.

Suitable Criteria for Culture

TRANSPORT TIME
Classification of sputum on the basis of leukocytes and
squamous epithelial cell densities SPECIMEN TIME RECOMMENDED
Respiratory (i.e sputum) 1 hour
Cell numbers per x100 (low power) Gastrointestinal (i.e stool) 1 hour
field Blood 1 hour
Group Leukocytes Epithelial CSF Immediately
cells Other body fluids Immediately
CEREBROSPINAL FLUID 6 <25 <25 Only sputum Urine 1 hour
• 1st tube – protein and glucose and special tests 5 >25 <10
samples in
Exudates and transudates 30 minutes
• 2nd tube – Gram stain and culture these
• 3rd tube – cell count and differential staining categories
4 >25 10-25 should be PLATING TECHNIQUE
cultured I. ISOLATION STREAK TECHNIQUE
EFFUSIONS 1. The inoculation loop is dragged across the surface
3 >25 >25
What and where to collect? of the agar back and forth in a zigzag motion until
2 10-25 >25
1. Synovial fluid – from joints approximately 30% of the plate has been covered
1 <10 >25
2. Pleural fluid – from pleural cavity (space between 2. Sterilize the loop and rotate the plate 90 degrees.
lungs and inner chest wall) Starting in the previously streaked section, the
3. Peritoneal fluid – from abdominal cavity
URINE loop is dragged through it two to three times
4. Pericardial fluid – from pericardial cavity Methods of Collection continuing the zigzag pattern.
5. Hydrocele – from testes 1. Midstream clean catch 3. The procedure is then repeated once more until
2. Cytoscopy or catheterization the fourth quadrant being cautious not to touch
UPPER RESPIRATORY TRACT 3. Suprapubic aspiration the previously streaked area in order to get
isolated colonies.
1. Nasopharyngeal swab
TRANSUDATES AND EXUDATES

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 3

 Microbiology Section (Part 1)

II. CONTINUOUS STREAKING TECHNIQUE (for urine only) 5. Incubate inverted plates and thioglycolate broth
1. Get the loopful of the urine aseptically by 36±1ºC for 18-24 hours
submerging vertically only the loop portion (shaft 6. Record the culture media plates/tubes used and date
not included). Inoculate the BAP by continuous and time of incubation in the worksheet.
streaking, spread the urine downward from the
first quadrant and streaking up to the 4th quadrant
without changing the loop.

CSF/EFFUSIONS
1. Label the plates and tube with the accession number
of the sample (ex. EX001)
PROCESSING, INOCULATION, STREAKING AND 2. About 1ml preferred (if more than 1 ml is submitted SPUTUM/ENDOTRACHEAL ASPIRATE
centrifugation is recommended). Use the sediment (do 1. Label the plates with the accession number of the
INCUBATION
not decant all fluid but leave at least 1 ml) for sample (ex. R001)
BLOOD (CONVENTIONAL METHOD) bacteriological investigation 2. Get a loopful of purulent part of the specimen to be
1. Label the Blood Culture Broth (BCB) with the accession 3. With a sterile Pasteur pipette, place one drop of CSF tested and make an evenly thin smear on a slide for
number of the sample (ex. BL001). Incubate for 18-24 onto the first quadrant of each plate (BAP, MAC, CA) Gram stain. Air dry. (Before sterilizing the loop, dip the
hours. and 3-5 drops in BHI broth. loop by rubbing in sand alcohol jar to clean the debris
2. Subculture from BCB is done as follows: 4. Streak with isolation into each agar plate. left on the loop)
a. Label the plates with laboratory number of the 5. Incubate inverted plates: BAP and CA, in 5-10% CO2 at 3. Using another sterilized loop, get another loopful of
sample (ex. BL001) 36±1ºC; MAC at 36±1ºC, ambient air for 18-24 hours purulent part of the specimen and inoculate each plate
b. Mix the blood sample by swirling the blood culture 6. Record the culture media plates/tubes used and the with similar size and quality of sample onto the center
bottle 2-3 times. date and time of inoculation in the worksheet. of the first quadrant.
c. By using a forceps, get a sterile cotton ball with 4. Streak with isolation onto each agar plate.
70% alcohol and sterilize the rubber stopper then 5. Incubate inverted plates: BAP, CA, GBA, and BCA in 5-
gently pass into the flame 10% CO2 at 36±1ºC; MAC at 36±1ºC, ambient air for
d. With a sterile disposable syringe and needle, 18-24 hours
aspirate at least 0.5mL blood from the bottle 6. Record the culture media plates used and the date and
e. Place a drop of the sample on each agar plate (BAP, time of inoculation in the worksheet.
CAP, and MAC)
f. Streak with isolation
3. Incubate: BAP and CA, in 5-10% CO2 at 36±1ºC
incubator; MAC at 36±1ºC incubator, ambient air for
18-24 hours.
4. Record the culture media plates used and the date and
time of inoculation in the worksheet
WOUND SWAB/DISCHARGE
5. If no growth, re-incubate the BCB until 7 days at 1. Label the plate plates and tube with the accession
36±1ºC, ambient air for further subculture number of the sample (ex. EX001)
2. Use 2 swabs. Using the first swab, roll the sides and tip
of the swab into the upper corner of the 1st quadrant
of BAP and MAC.
3. Submerge the swab in the thioglycollate broth then THROAT SWAB
aseptically break/cut the stick halfway 1. Label the plates and tube with the accession number of
4. Perform isolation streak technique onto the BAP and the sample (ex. R001).
MAC

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 4

 Microbiology Section (Part 1)

2. Roll the sides and tip of the swab onto the upper corner 3. Inoculate the swab onto BAP, MAC, SSA, and TCBS. After incubation, examine primary media plates (BAP,
of the 1st quadrant of BAP 4. Inoculate by rubbing the swab onto the center of the CA, and MAC) for growth
3. Perform isolation streak technique. first quadrant of one medium, changing sides of the
4. Incubate inverted plate in 5-10% CO2 at 36±1ºC for 18- swab each time to another medium. Do not discard the 1. GROWTH ON BAP ONLY
24 hours. swab and proceed to the next step.
5. record the culture media plates used and the date and 5. Dip the swab into the upper portion of the APW then
time of inoculation in the worksheet. into the Selenite F broth.
6. Streak the isolation onto each agar plate.
7. Incubate inverted platers: BAP, MAC, TCBS, and SSA
plates, at 36±1ºC in ambient air for 18-24 hours.
8. Incubate the Enrichment broth media:
a. At 36±1ºC in ambient air for 6 hours; or
b. If the processing of specimen was done in the late
office hours and no night duty, the enrichment
broth media should be subculture after and 2. GROWTH ON BAP AND MAC
overnight of 16-18 hours incubation at room
Compare BAP and MAC. Check if the colonies are the
temperature
same and presence of pigment on each medium. (if other
URINE 9. After incubation, subculture the enrichment broths
a. Label the plates (TCBS and SSA) with the specimen colonies are not the same, look also for other possible
1. label the plates and tube with the accession number of
number and the source (ex. SOO1 AP-TCBS, SOO1 pathogens specific for ABP).
the sample (ex. EX001)
2. mix thoroughly the urine specimen, if sample container SF-SSA).
is small, invert 3-4 times or swirl if the sample container b. For APW, aseptically get a loopful of broth at the
is large. surface and make an isolation streak into TCBS.
3. Get the loopful of the urine aseptically by submerging c. Incubate the plates at 36±1ºC for 16-24 hours.
vertically only the loop portion (shaft not included). d. For Selenite F, gently mix and get a loopful of
Inoculate the BAP by continuous streaking, spread the broth and make an isolation streak onto SSA.
urine downward from the first quadrant and streaking e. Incubate the plates at 36±1ºC for 16-24 hours.
up to the 4th quadrant without changing loop. 10. Record the culture media plates/tubes used and the
date and time of inoculation in the worksheet. 3. GROWTH ON CAP
4. Use another sterile loop and get loopful of urine to
inoculate MAC agar and do the isolation streak.
5. Incubate inverted plates at 36±1ºC for 18-24 hours.
6. Record the culture media plates used and the date and
time of inoculation in the worksheet.

4. NO GROWTH

WOUND DISCHARGE/SWAB
After incubation, examine the primary media plates
(BAP, MAC) and thioglycolate for growth.

STOOL/RECTAL SWAB 1. GROWTH ON BAP ONLY

1. Label the plates and tube with the accession number IDENTIFICATION AND WORKUPS OF SPECIMENS
of the sample (ex. S001)
2. Dip a swab into the stool sample. BLOOD

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 5

 Microbiology Section (Part 1)

1. GROWTH ON BAP AND MAC Compare BAP and MAC. Check if the colonies are the same
Compare BAP and MAC. Check if the colonies are the same and presence of pigment on each medium. (If other colonies
and presence of pigment on each medium. (if other colonies are not the same, look also for other possible pathogens
are not the same, look also for other possible pathogens specific for BAP).
specific for BAP).

1. GROWTH ON BAP AND MAC

Compare BAP and MAC. Check if the colonies are the
same and presence of pigment on each medium. (if other 3. NO GROWTH
colonies are not the same, look also for other possible
2. GROWTH ON CAP
pathogens specific for BAP).

SPUTUM/ENDOTRACHEAL ASPIRATE
After incubation, examine the plates (BAP, GBA, BCA,
and MAC) for growth.
3. WITH EVIDENCE OF GROWTH ON THIO BUT NO
GROWTH ON PRIMARY PLATES 1. GROWTH ON BAP ONLY

2. WITH EVIDENCE OF GROWTH ON THIO BUT NO

GROWTH ON PRIMARY PLATES
4. NO GROWTH ON PRIMARY PLATES AND THIO

3. NO GROWTH ON PRIMARY PLATES AND THIO

URINE
After incubation, examine the plates (BAP, MAC)
for growth. Quantitative growth on BAP by multiplying the
CSF/EFFUSIONS number of each colony type by 1,000 if 1 ul (0.001 mL) loop 2. GROWTH ON BAP AND MAC
After incubation, examine the primary media was used or by 100 if 10 ul (0.01 mL) loop was used Compare BAP and MAC. Check if the colonies are the same
plates (BAP, CAP and MAC) and TSB/BHI for growth. and presence of pigment on each medium. (if other colonies
1. GROWTH ON BAP ONLY are not the same, look for other possible pathogens specific
1. GROWTH ON BAP ONLY for BAP).

3. GROWTH ON BCA
2. GROWTH ON BAP AND MAC

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 6

 Microbiology Section (Part 1)

4. GROWTH ON BAP/GBA

Make a vertical streak then fishtail (zigzag) over slant

RESULTS AND INTERPRETATION

5. NO GROWTH LYSINE IRON AGAR (LIA)

- Used to determine whether a Gram-negative rod
decarboxylates or deaminates lysine
- LYSINE DEAMINATION occurs on the lysine slant
- LYSINE DECARBOXYLATION occurs in the lysine butt
STOOL
PROCEDURE
1. Observe for a typical colony on the following ulture
media after 18-24 hours of incubation:

SUSPECTED POSSIBLE
CULTURE MEDIA
COLONIES ORGANISMS TRIPLE SUGAR IRON (TSI)
White B- - Used to differentiate among the different groups of
Staphylococcus
BAP hemolytic/ non- Enterobacteriaceae
aureus, yeast
hemolytic colonies - Detects three primary characteristics of bacterium
Salmonella spp., 1. The ability of ferment sugars.
Non-lactose Shigella spp., 2. The ability to produce gas from the fermentation of
MAC fermenter or Aeromonas spp., sugars.
colorless (NLF) Plesiomonas spp., 3. Production of large amounts of hydrogen sulfide. Stab twice through the center of the butt up to the
E. coli
bottom of the tube then draw out and from the lower
Yellow or green PROCEDURE
TCBS Vibrio spp., portion of the slant, make a vertical streak then fishtail
flat colonies (Y/G)
over slant
NLF with or Salmonella spp.,
SSA RESULTS AND INTERPRETATION
without H2S Shigella spp.,

2. Perform biochemical tests/ serological typing on

suspected typical colonies on each medium

BIOCHEMICAL TESTS
IDENTIFICATION OF FERMENTERS AND NON-
FERMENTERS

CITRATE Stab through the center of the butt up to the bottom of

the tube then draw out and from the lower portion of SULFIDE-INDOLE-MOTILITY MEDIUM
- Used to determine the ability of bacteria to utilize
the slant, make a vertical streak then fishtail over slant. - A semisolid medium used as differential test medium
sodium citrate as its only carbon source and inorganic
RESULTS AND INTERPRETATION 1. SULFIDE – detection of the ability of an organism to
ammonium dihydrogen phosphate (NH4H2PO4) is the
liberate hydrogen sulfide (H2S) from sulfur bearing
sole fixed nitrogen source.
amino acids producing a visible, black color
reaction.
PROCEDURE

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 7

 Microbiology Section (Part 1)

2. INDOLE – to determine the ability of an organism to

split indole form the tryptophan molecule by
adding 3-5 drops of Ehrlich’s or Kovac’s reagent and
observe for development of a red color.
3. MOLITILITY – to determine if the organism is motile
or non-motile.
PROCEDURE Inoculate the broth with a heavy inoculum from 18-24
hour pure culture
RESULTS AND INTERPRETATION

3. By using a sterile applicator stick, pick a colony from

a colorless media and rub on the moistened filter
1. Stab through the center of the agar to a depth of 1/3 paper
to 1/2 of the medium making sure that the inoculating
needle was drawn out in the same stab

Indole: performed after incubation of the SIM

OXIDASE
- Determine the presence of bacterial cytochrome RESULTS AND INTERPRETATION
oxidase using the oxidation of substrate tetramethyl-p-
phenylene dihydrochloride to indophenol, a dark
purple-colored end product.
2. After incubation, add 5-7 drops of Ehrlich’s or Kovac’s - To separate Enterobacteriaceae from other bacteria like
reagent Vibrio, Neisseria species, Pseudomonas, Haemophilus,
RESULTS AND INTERPRETATION and other related bacterial species.
SULFIDE INDOLE MOTILITY PROCEDURE

O/F MALTOSE/DEXTROSE
- Tests the metabolism of sugar by prokaryotic cells.
1. Place a filter paper on a slide Cells may metabolize sugar in a variety of pathways,
and the OF test is studying whether sugar is
metabolized by aerobic respiration or by an anaerobic
respiration or by an anaerobic pathway including
UREASE fermentation.
- Used to determine if the microorganism that possesses - The OF medium has a low sugar and peptone content,
the enzyme urease that can hydrolyze urea, releasing and a high sugar content, making it a semi-solid
ammonia and producing a pink, red color change in the medium that is unlikely to go alkaline from protein
medium. utilization if the sugars are metabolized
PROCEDURE PROCEDURE

2. Dispense 1 drop of reagent onto the filter paper.

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 8

 Microbiology Section (Part 1)

Inoculate the test medium by stabbing the medium 3x PROCEDURE

RESULTS AND INTERPRETATION COAGULASE TEST
• Used to differentiate Staphylococcus aureus (positive)
from other Staphylococcus spp. (negative) – coagulase
negative Staphylococcus (CoNS).
• Coagulase is an enzyme produce by S. aureus that
converts (soluble) fibrinogen in plasma to (insoluble
fibrin)

SLIDE COAGULASE TEST

IDENTIFICATION OF STAPHYLOCOCCUS SPP. - Screening detects clumping factor (bound
Staphylococcus sp. Place 2 separate drops of hydrogen peroxide coagulase)
• Gram positive bacteria - Read results in 10 seconds
• Round/cocci - Result:
• Grape-like clusters POSITIVE Presence of white precipitate or
• Non-spore forming bacteria agglutination within 10-15 seconds
• Facultative anaerobes NEGATIVE Smooth and milky/homogenous
• Heat-resistant organism mixture
• Can tolerate high salt content media

IDENTIFICATION OF STAPHYLOCOCCUS SP.

1. Describe the colony morphology on BAP
• Size
• Color
• Hemolysis (Beta or Gamma)
2. Gram stain
• Gram-positive cocci
• Form clusters “grape-like”
Pick a colony from an 18–24-hour culture and immerse
• Occurs singly, in pairs, tetrads, short chains
into the H2O2. Do not mix.
CATALASE TEST
- Used to differentiate staphylococcus (+) from PROCEDURE: SLIDE GOAGULASE
streptococcus (-)
- Reagent: hydrogen peroxidase (H2O2) – catalase
mediates the breakdown of H2O2 to hydrogen and
water
- Result:
POSITIVE Immediate bubbling or
effervescence
NEGATIVE No bubbling formed

Place a 2 drops of reconstituted coagulase plasma on a

clean glass slide

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 9

 Microbiology Section (Part 1)

LABORATORY IDENTIFICATION OF STREPTOCOCCUS

SP.
1. Bacitracin Susceptibility Test
2. Optochin Susceptibility Test
3. CAMP Test
4. Bile Esculin
Incubate at 36ºC for 2-4 hours and observe for a clot 5. 6.5% NaCl
formation by gently tilting the tube.
If no clot is observed, re-incubate the tube and read BACITRACIN SUSCEPTIBILITY TEST
after 24 hours.
- Used to distinguish Group A streptococci, from other
Flow diagram on identification of Staphylococcus spp. streptococci. This test is used to determine the effect
Pick a colony of the test isolate and emulsify into the of small amount of bacitracin (0.04U) on an organism.
drop of coagulase plasma - Result:
SUSCEPTIBLE Any zone of inhibition around the disk
TUBE COAGULASE TEST RESISTANT No zone of inhibition
- Definitive
- Detects free coagulase
- Reagents: commercially prepared Rabbit’s
plasma/dehydrates plasma containing citrate or
EDTA
- Examine tube after: 2 hours, 4 hours, and 24
hours of incubation
- Result:
POSITIVE Any degree of clotting/coagulum
Positive
NEGATIVE No clot/coagulum formation

Streptococcus pyogenes is Bacitracin-SUSCEPTIBLE

IDENTIFICATION OF STREPTOCOCCUS/
Negative ENTEROCOCCUS PROCEDURE
Streptococcus sp.
• Gram-positive cocci in pairs and in chains
• Nonmotile, non-spore forming
• Facultatively anaerobic
• Catalase negative
• BAP and CAP
• 35ºC in 5-10% CO2 atmosphere
• Blood, CSF, Upper Respiratory Tract, Lower
PROCEDURE: TUBE COAGULASE Respiratory Tract, Exudates

Streptococcus Classification
• Hemolytic reaction on SBAP
• Physiologic – pyogenic, viridans, lactic and
enterococcal
• Lancefield – Group A-H, L-O
Emulsify 1-3 colonies of the isolate in a tube containing
0.5mL of coagulase plasma

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 Microbiology Section (Part 1)

Create a three-layered on half of the BAP with a sterile <9 mm Resistant

loop containing a few colonies of the test isolate

Incubate the plate for 18-24 hours at 36ºC, 5-10% CO2

(hindi kita sa vid, pero sinidihan yung candle tapos
nilagay sa loob ng Tupperware the tinakpan)

CAMP TEST
PROCEDURE - Christie, Atkins, Munch-Peterson
- Identify group B β-hemolytic streptococci
Make sure that the lawn is formed side by side (Steptococcus agalactiae) based on their information
streaking to ensure confluent growth of a substance (CAMP factor) that enlarges the area of
hemolysis formed by the β-hemolysin elaborated from
Staphylococcus aureus.
- Result:
With a sterile loop, pick a single a-hemolytic colony POSITIVE Arrow-shaped zone of hemolysis
then create a three-layered lawn on half of the BAP NEGATIVE No arrow-shaped hemolysis formed

With sterile forceps, place a bacitracin disk on the lawn.

Gently, press the disk so that it adheres to the agar
surface.

Make sure that the lawn is formed side by side

streaking to ensure confluent growth
PROCEDURE

Incubate the plate for 18-24 hours at 36ºC, 5-10% CO2

(hindi kita sa vid, pero sinidihan yung candle tapos
nilagay sa loob ng Tupperware the tinakpan)

OPTOCHIN SUSCEPTIBILITY TEST

Make a vertical line using a sterile loop with a few
- Used in the presumptive identification of alpha- colonies if S. aureus ATCC 25923 on BAP
hemolytic Streptococcus pneumoniae, which is
optochin sensitive
- Result:
ZONE OF INHIBITION INTERPRETATION With sterile forceps, place a bacitracin disk on the lawn.
≥14 mm Susceptible Gently, press the disk so that it adheres to the agar
9 – 13 mm Intermediate surface.

CAMILLE GRACE A. ESPALLARDO | BSMT 4-3 11

 Microbiology Section (Part 1)

Draw a perpendicular line of the test isolate about 4-6 PROCEDURE

mm away from the S. aureus ATCC 25923 line

BILE ESCULIN TEST (-) (+)

Tests the ability of organisms to hydrolyze esculin in the
presence of bile. It is commonly used to identify members
of the genus Enterococcus (E. faecalis and E. faecium).
Result:
Diffuse blackening of more than half of
POSITIVE
the slant within 24-48 hours
NEGATIVE No blackening
Stab the tubed medium 5-8 times with 5-10 colonies of
the test isolate.

PROCEDURE IDENTIFICATION OF HAEMOPHILUS INFLUENZAE

AND NEISSERIA GONORRHEA

HAEMOPHILUS INFLUENZAE
• Large, colorless to gray colonies
• No discoloration of chocolate agar plate
• With pungent odor
• Gram-negative coccobacilli
PROCEDURE • Pleomorphic

Emulsify 2-3 colonies of the tests isolate into 6.5% NaCl

broth.

ARABINOSE FERMENTATION TEST

- Differentiate Enterococcus faecium (+) from
Growth requirements
Enterococcus faecalis (-)
- Result: 1. X factor or hemin
Inoculate the bile esculin medium with 2-3 colonies of POSITIVE yellow 2. V factor or NAD (nicotinamide adenine
the test isolate with an inoculating needle. dinucleotide)
NEGATIVE No color change

IDENTIFICATION OF HAEMOPHILUS INFLUENZAE

6.5% NACL
1. X and V factor Dependence test
- Tests the ability of organisms to grow in high
2. Satellitism/Hemolysis Test
concentrations of salt
- Result:
Turbidity or presence of obvious X AND V TEST
POSITIVE - Determines nutrient requirement of organisms
bacterial growth in the medium
NEGATIVE Clear or no growth 1. Prepare turbid suspension (~1.0 MacFarland)
2. Inoculate on trypticase soy agar plate.
3. Place disks X, V, and XV

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 Microbiology Section (Part 1)

4. Incubate in CO2 +35 to +37ºC Notes from the synchronous session of Ms. Gracia M.
Catulin, RMT
EACMC-Cavite video presentation for alternative medical
technology internship program: Microbiology section

NEISSERIA GONORRHEAE
Factor requirement • Grows on BAP (non-hemolytic) and CAP
Haemophilus species Β-hemolysis • G (-) diplococci: coffee bean-shaped
X V
H. influenzae + + - • Can survive either as an extracellular organism, or,
H. parainfluenzae - + - alternatively, as an intracellular organism
H. haemolyticus + + +
H. parahaemolyticus - + +

SATELLITISM/HEMOLYSIS TEST
- When grown in blood culture media, S. aureus
produce NAD as a metabolic by product. For that
season, species of Haemophilus may grow very closely
to the colonies of S. aureus when streaked on sheep
blood agar
- S. aureus produces NAD (V factor), a key requirement
for the growth of H. influenzae. This phenomenon is
IDENTIFICATION OF NEISSERIA GONORRHEAE
known as satelliting, and the test is called satellitism.
• Oxidation test
1. Using the same suspension for the X and V growth • Superoxol test
requirement test, dip a sterile swab carefully into
the bacterial suspension and streak in a close OXIDATION TEST
zigzag motion of BAP. Allow to dry. - Determines the presence of bacterial cytochrome
2. Streak vertical line of Staphylococcus aureus on oxidase using the oxidation of the substrate
the middle of the zigzag pattern and incubate in a tetramethyl-p-phenylene dihydrochloride to
5-10% CO2 incubator for 18-24 hours at 36+ºC indophenol, dark purple-colored end product
3. Interpret as follows: - N. gonorrhea: positive
Presence of growth with β-hemolysis
POSITIVE SUPEROXOL TEST
near or around the S. aureus streak
NEGATIVE Presence of growth without β- - Catalase
hemolysis - 30% hydrogen peroxidase
- Positive: effervescence
- Negative: little to no effervescence

REFERENCES
Notes from the discussion of Ms. Gracia M. Catulin, RMT

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 MICROBIOLOGY SECTION (PART 2)

STAINING METHODS GRAM STAIN GRAM STAIN MORPHOLOGY

POSITIVE STAINING - A differential staining technique based on bacterial cell
- Actual cells are stained and appear in a clear background wall structure
- Classified bacteria as:
1. SIMPLE STAIN o GRAM-POSITIVE
o A stain which provides color contrast but gives same o GRAM-NEGATIVE
color to all bacteria and cells
GRAM STAIN REAGENT
2. DIFFERENTIAL STAIN
o A stain which imparts different colors to different
bacterial cells GRAM STAIN - DIRECT FROM SAMPLES
RESPIRATORY/SPUTUM SAMPLES
Suitability Criteria of Sputum for Culture

GRAM STAINING RULES

ALL Cocci are Gram Positive Except:
- Neisseria
EXUDATES/URINE/TISSUE
- Veillonella
NEGATIVE STAINING
- Moraxella
- The background is colored to create a contrast to aid in Pus cells, Epithelial cells/LPF
*NVM - NeVer Mind
the better visualization of cellular structures <1/LPF 1+ RARE
ALL Bacilli are Gram Negative Except: 1-10/LPF 2+ FEW
- Bacillus 11-25/LPF 3+ MODERATE
- Bifidobacterium >25/LPF 4+ MANY
- Actinomyces
- Nocardia Organisms/OIF
- Streptomyces <1/OIF 1+ RARE
1-10/OIF 2+ FEW
- Clostridium
11-25/OIF 3+ MODERATE
- Corynebacterium
>25/OIF 4+ MANY
- Erysipelothrix
- Listeria
Indicate the absence of cells as “NONE” and organisms as
INDIA INK - Lactobacillus
“NO ORGANISM SEEN”

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 Microbiology Section (Part 2)

GRAM STAINING PROCEDURE (URINE SAMPLE)

Step 1. Prepare a slide Step 9. Flood slide
Step 3. Using the edge
smear. Smear a very with safranin and let it
of a cover slip, mix.
thin layer onto the stand for 1 minute
slide, enough to dry
completely within a
few seconds
Step 4. Cover entire
Step 2. Air dry and Step 10. Wash gently
smear with the cover
heat fix by passing the with water. Air dry.
slip
slide 2-3 time over a
flame and allow to
Step 11. Examine under low power objective and
cool
estimate the number of SEC and leukocytes
Step 12. Examine under OIO to determine predominant Step 5. Let it stand for
Step 3. Flood the and other organisms present 5 minutes
smear with crystal
violet and let stand for
1 minute GRAM STAIN - QUALITY CONTROL
- Controls should be run on a daily basis
- Standard control depends on the manufacturer, as long Step 6. Look for white
as Gram positive is recognizable from Gram negative cells in LPF the shift to
Step 4. Wash gently
HPF
with water. Pour off
excess water GRAM STAIN - IMPORTANT POINTS
- Use fresh/young cultures: must be within 18-24 hours old INDIA INK RESULTS
- Thin, thick or uneven smears will result in poor/uneven
staining and decolorization Findings Reporting
Step 5. Flood smear Encapsulated yeast cells Positive for
- Do not heat-fix for very long
with gram’s iodine and – present encapsulated yeast
- Perform quality control of reagents
let stand for 1 minute cell/s
Encapsulated yeast cells No encapsulated yeast
INDIA INK – absent cell/s seen
- Staining of cerebrospinal fluid to test for the presence of
Step 6. Wash gently Cryptococcus spp.
with water. Pour off ACID FAST BACILLI STAINING
excess water - Acid-fast bacilli: rod-shaped bacilli that can be seen
INDIA INK STAINING PROCEDURE
under the microscope following a staining procedure in
Step 7. Decolorize with which the bacteria retain the color of the stain after an
acetone-alcohol 5 to Step 1. Place a drop of acid wash
10 seconds until the india ink in a clean
alcohol runs most glass slide
DIRECT SPUTUM SMEAR MICROSCOPY (DSSM)
clear (Be careful not to
- Involves the examination of a series of sputum specimens
over-decolorize)
from each patient and requires repeated patient visits to
Step 2. Add a drop of health facilities to submit specimens and to collect
Step 8. Immediately sample to the india ink - Screening for pulmonary tuberculosis
wash with water

GAIL YVANA D. SARMIENTO | BSMT 4-3 2

 Microbiology Section (Part 2)

PULMONARY SPECIMEN (2 SAMPLES) Step 4. Air dry and

1. Spot-early morning collection heat fix by passing Step 12. Gently
2. Spot-spot collection (at least one hour apart) the slide 2-3 times wash with tap
over a flame and water and tip to
allow to cool drain excess water
SMEAR CHECK POINTS
1. Sputum quality DSSM RESULTS
2. Staining Step 5. Place fixed
slide on the staining Reporting AFB Seen
3. Cleanness
rack Scale
4. Thickness
0 No AFB seen in 300 visual fields (VF)
5. Size
+n 1-9 AFB seen in 100VF (write the actual
6. Evenness # of AFB seen, e.g. +1, +2, … +9)
Step 6. Pour carbol
NOTE! Stained smear has 2 cm width and 3cm length fuchsin on the 1+ 10-99 AFB seen in 100 VF
smear covering 2+ 1-10 AFB / OIF in at least 50 VF
entirely 3+ More than 10 AFB / OIF in at least 20
VF
Step 7. Apply
Visual Field
enough heat
underneath until
steam comes of
from the stain. (Do
DSSM PROCEDURE not boil or dry out)

Step 8. Leave it for

Step 1. Label the OTHER TESTS
10 minutes
slide POTASSIUM HYDROXIDE (KOH) MOUNT
- Used for the rapid detection of fungal
Step 9. Gently rinse elements in clinical specimen, as it clears
Step 2. Using a with tap water to the specimen making fungal elements more
wooden applicator remove excess visible during direct microscopic
stick, pick the carbol fuchsin. At examination
purulent particles this point, the - Reagent: 10% KOH
of the specimen smear is red in color
Step 3. Spread the Step 10. Decolorize PROCEDURE
specimen evenly with acid alcohol
unto the center of until pink color
the slide by making disappears from the Step 1. Place 1-2 drops
a small, circular, smear of 10% KOH on a clear
coil-type pattern glass slide
smear. Step 11.
Counterstain with
Spreading should methylene blue for
2-3 seconds. Step 2. Add a small
start on the
amount of specimen in
innermost center to
10 % KOH
the periphery /
border of the smear

GAIL YVANA D. SARMIENTO | BSMT 4-3 3

 Microbiology Section (Part 2)

Step 2. Emulsify a
Step 3. Mix the KOH loopful of growth from Step 3. Incubate the
and specimen using a colony to the drop of suspension at 35-37oC
the edge of the cover 3% KOH. Stir the for 2-3 hours
slip. suspension
continuously for 60
seconds. Step 4. After
incubation, place a
Step 4. Cover the drops
drop of suspension on
with the cover slip Step 3. Gently pull a clean glass slide and
away the loop from the cover with a cover slip
suspension

Step 5. Wait for 5 Step 5. Examine under

minutes for cellular KOH STRING TEST RESULT
low & high-power
clearing POSITIVE Organisms become thick, stringy and objective
form long strands with the first 30 sec.
This is seen in GRAM NEGATIVE
BACTERIA GERM TUBE RESULTS
Step 6. Examine under
NEGATIVE Organisms leave the suspension POSITIVE: a short hyphal
the microscope with a
unaltered or absence of stringing. This (filamentous) extension
reduced light source at
is seen in GRAM POSITIVE BACTERIA arising laterally from a
LPO.
yeast cell, with no
constriction at the point
POTASSIUM HYDROXIDE (KOH) MOUNT RESULTS of origin. Germ tube is
Observe for the following structures: half the width and 3-4
1. Yeast cells (budding or single cells) times the length of the
2. Psuedohyphae yeast cell and there is no
3. Hyphal elements: maybe hyaline (light or no presence of nucleus
pigment) or dematiaceous (dark brown) GERM TUBE NEGATIVE: no hyphal
Reporting (filamentous) extension
- An outgrowth produced by spores of spore-releasing
POSITIVE Report as “positive for (fungal arising from a yeast cell or
fungi during germination. The germ tube differentiates,
elements observed)” ex. yeast and a short hyphal extension
grows, and develops by mitosis to create somatic hyphae
hyphal elements. constricted at the point of
NEGATIVE Report as “No fungal elements found” origin
PROCEDURE
SEROTYPING
KOH STRING TEST Step 1. Place 0.5 mL of - Serotypes are groups within a single species of
- Relies on the differential resistance to 3% potassium fresh serum in clean microorganism, such as bacteria or viruses, which share
hydroxide between gram positive and negative cells test tube distinctive surface structures

PROCEDURE PROCEDURE
Step 2. Pick a colony 1. Place a drop of each of anti-sera on a clean glass slide
then emulsify in the 2. Using a sterile applicator stick, pick several colonies form
Step 1. Place a drop of
fresh serum the plate and emulsify on each drop, creating
3% KOH on a clean
glass slide homogenous, slightly milky suspensions

GAIL YVANA D. SARMIENTO | BSMT 4-3 4

 Microbiology Section (Part 2)

3. Mis the anti-serum with the suspension using a sterile 2. Gradient diffusion method ANTIBIOTIC DISKS
applicator stick 3. Automated antimicrobial susceptibility testing - Stock antibiotic disks should be stored at -20oC
4. Tilt the glass slide back and forth for 1 minute and system - Working antibiotic disks should be stored at 4oC
observe for agglutination - Antibiotic disks used are based on Clinical and Laboratory
5. Interpret as follows: DISK DIFFUSION METHOD Standards Institute (CLSI) panels
POSITIVE Strong agglutination appears within 30 - Antibiotic paper disks are placed on agar medium surface
seconds to 1 minutes inoculated with the test organism Disks per plate guide
NEGATIVE Homogenous suspension - Kirby-Bauer Method Maximum # of Disks
*Agglutination is grossly observed with light passing 140 mm or 100 mm or 90
through the slide. Delayed or weak agglutination is Organisms
150 mm (big mm (small
regarded as negative plate) plate)
Enterobacteriaceae
Pseudomonas
aeruginosa 12 5
Acinetobacter spp.
Enterococcus spp.
Haemophilus spp. 9 4
Streptococcus
pneumoniae
MEDIUM FOR DISK DIFFUSION Streptococcus spp.
Agar for AST 9 4
Viridans group
✓ pH 7.2-7.4 Streptococcus spp.
ANTIMICROBIAL SUSCEPTIBILITY TESTING ✓ Low Thymine and Thymidine β hemolytic group
- Measures the ability of microbial agent/s to inhibit in ✓ Sufficient divalent Cation (Ca++ and Mg++) Neisseria
9 3
vitro bacterial growth gonorrhoeae
- Indicated for an etiologic agent of an infection that needs Mueller Hinton Agar Neisseria
5 2
chemotherapy ✓ Good batch to batch reproducibility meningitidis
- Guide the clinician in selecting the best and appropriate ✓ Low in antibiotic inhibitors against sulfonamides
antimicrobial agent (to predict the outcome of treatment trimethoprim, tetracycline INOCULATION OF AGAR PLATES
with the antimicrobial tested) ✓ Supports the growth of most non - fastidious - Use appropriate check plate for a particular isolate
pathogens Organism Check Plate
METHODS Gram positive
AST Agars Staphylococcus spp.
1. Disk diffusion BAP
Organisms Agar Plate Streptococcus spp.
2. Dilution method
Gram-negative organisms Mueller – Hinton Agar Neisseria meningitidis
3. Gradient diffusion method Gram-negative MAC
Staphylococcus spp. (MHA)
4. Automated antimicrobial susceptibility testing Fastidious
Enterococcus spp.
system Haemophilus spp. CAP
Streptococcus spp. Mueller – Hinton Agar
Neisseria meningitidis (MHA) w/ 5% sheep’s Neisseria gonorrhoeae
MINIMAL INHIBITORY CONCENTRATION blood
- Lowest concentration of an antimicrobial agent that Haemophilus spp. Haemophilus test medium INCUBATION GUIDE
would inhibit visible in vitro growth of a test organism (HTM) - Inverted plates are incubated at the desired
over a defined interval related to the organism’s growth Neisseria gonorrheae GC agar temperature, atmosphere, and duration of incubation
rate depending on the test organism’s requirements
- Determined by:
1. Dilution method

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 Microbiology Section (Part 2)

Organism Agar Temp Atmos Time Step 4. Dip the loop Step 11. Apply
Enterobacteriaceae into NSS and emulsify appropriate antibiotic
16 –
Pseudomonas on the inside wall of disks manually by using
20 h
aeruginosa the tube for equal sterile forceps onto the
Acinetobacter spp. distribution of the inoculated agar surface
Burkholderia spp. 20 – organism with minimum spacing
Stenotrophomas 24 h of 24 mm center to
MHA - center between disks
maltophilia Step 5. Mix the
Other non – 16 – suspension by tapping
35oC Gently press each disk
Enterobacteriaceae 20 h or inverting the tube
±2 down with sterile
Staphylococcus
24 h forceps for every
spp. Step 6. Using a
Enterococcus spp. application of the disk
densitometer, adjust
MHA the bacterial
Streptococcus spp. w/ suspension to the READING ZONES
5% 20 –
β hemolytic grp 5% required 0.5 McFarland - Using calipers or ruler measure the
CO2 24 h
Viridans grp sheep standard diameter of the complete zone of
blood Step 7. Dip a sterile inhibition
cotton swab into the - Read MHA plate with unaided eye
15-MINUTE RULES standardized bacterial using transmitted light
Within 15 minutes: suspension, and - On MHB, GC agar and HTM, remove
✓ After preparing inoculum, seed the agar express excess fluid the cover and measure inhibition
✓ Apply disk on seeded agar against the inside wall zones from the surface illuminated
of the tube
✓ After disk application, incubate plate with reflected light
Step 8. Swab the entire
surface of the AST
DISK DIFFUSION PROCEDURE 1. Assessing Growth
medium 3 times,
rotating the plate
through an angle of Read plates only when the lawn
Step 1. Label the MH
about 60oC after each growth is confluent (A)
plate with the
application and pass
specimen accession no.
the swab around the
rim of the agar surface
Step 9. With the same Repeat the test when individual
Step 2. Prepare 2-3 mL swab, inoculate by colonies are apparent (B)
sterile NSS in sterile 5 touching/rubbing the
mL round bottom tube check plate, discard
the swab accordingly 2. Measuring the zones of inhibition
Step 3. Using a sterile
inoculating loop, select Step 10. Streak the
and pick 3-5 similar inoculum in the check Measure zones of inhibition to
isolated colonies by plate with sterile the nearest whole millimeter
touching the top of the inoculating loop (mm)
colonies

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 Microbiology Section (Part 2)

TEST RECOMMENDATIONS FOR DIFFERENT

ORGANISMS
Measure growth with no zone of
inhibition as 6 mm
I. DIRECT β-LACTAMASE TEST (NITROCEDIN DISK)
1. Enterococcus spp.
2. Haemophilus influenzae/parainfluenzae
- Zones of complete inhibition include the diameter of 3. Moraxella catarrhalis
the disk and show no obvious, visible growth as judged 4. Neisseria gonorrhoeae
by the unaided eye - Double zones 5. Neisseria meningitidis
- Measure the sone of growth inhibition, not the sone of 1. When double zones are observed, check the 6. Staphylococcus spp.
hemolysis. Tilt the plate to better differentiate growth for purity and repeat the test, if 7. Pasteurella spp.
between hemolysis and growth necessary.
- Discrete colonies growing within the zone: 2. If the culture is pure, measure the inner zone II. SCREENING FOR STAPHYLOCOCCUS SPP.
1. When discrete colonies grow within a clear
1. Direct β-lactamase test
zone of inhibition, repeat the test with a pure
2. Mec-A-mediated oxacillin resistance using
culture or subculture of a single colony from
cefoxitin
the primary culture plate
3. Inducible clindamycin resistance
2. When the discrete colonies continue to grow
within the zone of inhibition after repeating the
III. FOR INDUCIBLE CLINDAMYCIN RESISTANCE (ICR)
test, measure the colony-free inner zone
1. Staphylococcus spp.
2. Β-hemolytic Staphylococcus spp.
QUALITY CONTROL 3. Streptococcus pneumoniae
- Antibiotics should be regularly tested if they still function
properly TEST RECOMMENDATIONS FOR
- Guide to which antibiotic to test, with what organism, ENTEROBACTERIACEAE
- Measuring zone of inhibition using transmitted light and their range are indicated in the CLSI Document no. 1. Extended Spectrum β-lactamase Test (ESBL)
1. For some tests (e.g., linezolid against M100 1. E. coli, Kleb. spp., P. morabilis
Staphylococcus spp.) measure zone of - Control strains must be used as reference: 2. AmpC β-lactamase screening test
complete inhibition with transmitted light 1. Staphylococcus aureus 25923 - Not yest recommended by CLSI
2. Position the inverted petri plate in front of a 2. Escherichia coli 25922 3. Test for diminished fluoroquinolone susceptibility
light source for zone examination 3. Pseudomonas aeruginosa 27853 testing using pefloxacin (extraintestinal Salmonella)
- Trimethoprim-sulfamethoxazole 4. Streptococcus pneumoniae 49619 4. Carbapenemase screening test
1. When measuring the zone of inhibition for 5. Haemophilus influenzae 49247 5. Metallo-β-lactamase test (MBL)
Trimethoprim-sulfamethoxazole, disregard 6. Escherichia coli 35218
slight or hazy growth (20% or less of the lawn
METHICILLIN-RESISTANT STAPHYLOCOCCUS
growth) and measure the obvious zone margin DETECTION OF MULTIPLE DRUG-RESISTANT AUREUS (MRSA)
ORGANISMS (MDRO) - mecA or mecC-mediated oxacillin resistance in
MULTIDRUG RESISTANT ORGANISMS (MDRO) staphylococci
- Bacteria that have become resistant to more than one - disk diffusion: CEFOXITIN as surrogate antibiotic
class of antimicrobial agents - medium:
- Swarming - Usually are resistant to all but one or two commercially 1. mueller hinton agar w/ 4% NaCl for oxacillin only
1. Strains of proteus spp. May swarm into areas of available antimicrobial agents, complicating treatment of 2. mueller hinton agar w/ot NaCl for cefoxitin and
inhibited growth. Ignore the thin veil of illness they cause other antibiotics
swarming growth in an otherwise obvious zone
of inhibition

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 Microbiology Section (Part 2)

- Procedure: Note: Routine screening of Proteus mirabilis for ESBL

1. following the standard disk diffusion, place β- Penicillin production is not recommended. However, when it is
Penicillin Penicillin
cefoxitin disk onto plain mueller hinton agar. lactamase disk zone deemed clinically relevant, (ex. Bacteremic isolate) screen
disk report
(MIC: oxacillin onto mueller hinton agar w/ 2-4% test report edge test for ESBL
NaCl) R (+) Sharp R
2. incubate plain MHA (cefoxitin) for 16-20 hrs S - Can be transferred to enteric bacilli (Samonella, Shigella,
(-) Fuzzy S
3. read susceptibility results >29mm Citrobacter fruendii, and Serratia marcscens)
4. note: for oxacillin (and vancomycin), read with S - Appear to be susceptible to certain antibiotics
(+) Sharp R
transmitted light; for cefoxitin (and other >29mm - ESBL producing isolates are susceptible to beta-
antibiotics), read with reflected light lactam/beta-lactamase inhibitor combination agents in
5. record and interpret according to the latest CLSI D-TEST: INDUCIBLE CLINDAMYCIN RESISTANCE (ICR) vitro but the effectiveness is uncertain
document. - A disk diffusion test using clindamycin and erythromycin e. g., clavulanic acid, sulbactam, and tazobactam – inhibit
mecA-mediated Cefoxitin Oxacillin Oxacillin disks placed in close proximity to detect the presence of the ESBLs produced by the organisms when tested with
interpretation
resistance disk MIC report inducible clindamycin resistance in staphylococci and
mecA- cefotaxime, ceftazifime, cefepime, or aztreonam
R (or streptococci
mediated
R R do not MRSA DOUBLE DISK DIFFUSION METHOD
(or the rare
report) PROCEDURE
mecC) - Detection of ESBL is by
mecA- 1. Following the general procedure for antimicrobial using disk containing
homologues MRS susceptibility, place clindamycin 2ug disk *15-25 mm clavulanic acid (AMC)
R (or
(e.g., (other (Staphylococcus spp.) apart from the edge of and placing it between
R R do not
mecA1, CoNS erythromycin 15ug disk on susceptibility plate.
report) cefotaxime (CTX) and
mecA2, spp.) *Beta-hemolytic Streptococcus spp. (12-15 mm. disk aztreonam (ATM) disk
mecC1) distance - An elliptical clearing of
Not *Streptococcus pneumoniae (12-15 mm. disk distance)
(-) S R R MRSA / distortion of inhibition
2. Incubate plates for 16-24 hours between the 2 disks
MRS 3. Read susceptibility test results and observe presence
MSSA / indicates inhibition of β-lactamases by clavulanic acid
(-) S S S of ICR - Screening test
MSS

POSITIVE Flattening (D-zone) of clindamycin zone

PENICILLIN DISK ZONE EDGE TEST FOR β- AmpC β-LACTAMASES
adjacent to erythromycin disk or hazy
LACTAMASE PRODUCTION OF STAPHYLOCOCCUS - Chromosomal or
growth inside the zone of inhibition (no D-
SPP. plasmid-encoded
zone) adjacent to erythromycin disk
- Performed on mueller-hinton agar using a 10-U penicillin enzymes. Isolates that
NEGATIVE No flattening or hazy growth of
disk following CLSI guidelines clindamycin zone adjacent to produce AmpC
- After 16 to 18 hrs of incubation in ambient air, a sharp erythromycin disk enzymes may have an
zone edge was interpreted as positive and a fuzzy zone antimicrobial
as negative for β-lactamase production EXTENDED SPECTRUM β-LACTAMASES susceptibility profile
- Plasmid-mediated enzymes produced by similar to those that
Enterobacteriaceae derived from mutations of TEM-1, produce ESBLs in that
TEM-2, and SHV-1 they show reduced
- Capable of hydrolyzing extended spectrum susceptibility to penicillin, cephalosporins, and
cephalosphorins, penicillins, and aztreonam aztreonam
- Most often associated with: - Found in enterobacter spp., K. aerogenes, Citrobacter
1. Escherichia coli spp., Serratia spp., and some other gram-negative
2. Klebsiella spp. species

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 Microbiology Section (Part 2)

PROCEDURE: 6. Inoculate an MHA plate with E. coli ATCC 25922 as for the REPORTING OF CULTURE RESULTS
1. Following the general procedure for antimicrobial routine disk diffusion procedure making the inoculum BLOOD
susceptibility testing, place cefotaxime (30ug) disk 15-25 suspension preparation and MHA plate inoculation steps 1. Preliminary Report
mm apart from edge of imipenem 10ug disk on the are each completed within 12 minutes. Allow the plates - The preliminary report for the absence of growth
susceptibility plate to dry for 3-10 minutes before adding the meropenem should be reported as: “NO GROWTH AFTER 24
2. Incubate plates for 16-25 hrs disks. HOURS OF INCUBATION”
3. Read susceptibility test results and observe presence of 7. Remove the meropenem disk from each TSB-meropenem
phenotypic resistance disk suspension using a 10-uL loop by placing the flat side 2. Final Report
POSITIVE Flattened edge of the inhibitory zone of the loop against the flat edge of the disk using a surface - Final report with the presence of growth should
around cefotaxime disk adjacent to an tension to pull the disk out of the liquid. Carefully drag be reported as: “POSITIVE FOR (IDENTIFIED
imipenem disk and press the loop along the inside edge of the tube to ORGANISM) AFTER (HOURS/DAYS) OF
NEGATIVE No flattened edge of the inhibitory zone expel excess liquid from the disk. Continue using the loop INCUBATION”. (Susceptibility test results are
around cefotaxime disk adjacent to an to remove the disk from the tube and then place it on the included if applicable)
imipenem disk MHA place previously inoculated with the meropenem - Final report with negative result for any organism
susceptible E. coli ATCC 255922 indicator strain. Disk should be reported as “NO GROWTH AFTER 7
CARBAPENEMASE capacity: 4 disks on a 100mm MHA plate; 8 disks on a 15- DAYS OF INCUBATION”.
- Beta-lactamase with versatile hydrolytic capacities. They mm MHA plate
have the ability to hydrolyze penicillins, cephalosporins, 8. Invert and incubate the MHA plate at 35oC ± 2oC in 3. Clinical significance
monobactams and carbapenems. Bacteria producing ambient air for 18-24 hours - If a typical pathogen is found from blood culture
these beta-lactamases may cause serious infections in 9. Following incubation measure the zones of inhibition as it is almost always significant. But many bacteria
which the carbapenemase activity renders many beta- for the routine disk diffusion method are often significant but may occur as
lactams ineffective. 10. Interpret as follows: contaminants in blood culture. Therefore, the
- Carbapenemase activity in Enterobacteriaceae and P. a. Carbapenemase positive: finding maybe be discussed with clinician or place
aeruginosa can be confirmed using CarbaNP colorimetric i. Zone diameter of 6-15mm or presence of a note in the result form “Please correlate
microtube assay or the modified carbapenem pinpoint colonies within a 16-18mm zone clinically”
inactivation method (mCIM) test. ii. If the test isolate produces a carbapenemase,
- Both the CarbaNP and mCIM tests may detect the meropenem in the disk will be hydrolyzed Record both preliminary and the final report and the date
carbapenemase production, but neither of these tests and there will be o inhibition of limited growth of release in the designated logbook.
can identify which carbapenemase is present inhibition of the meropenem-susceptible E. coli
ATCC 95922 CSF / EFFUSIONS
PROCEDURE b. Carbapenemase negative: CULTURE EXAMINATIONS
i. Zone diameter of ≥ 19 mm (clear zone) - Absence of colonies on the primary plates (direct
1. For each isolate to be tested, emulsify a 1-uL loopful of
ii. If the test isolate does not produce inoculation of specimen onto agar plates) and BHI broth
bacteria for enterobacteriaceae of 10-uL loopful of
carbapenemase, the meropenem in the disk has no sign of growth, record and report. Primary plates
bacteria for P. aeruginosa from an overnight blood agar
will not be hydrolyzed and will inhibit growth of and BHI broths are inspected everyday for the sign of
plate in 2mL TSB
meropenem-susceptible E. coli ATCC 95922 growth.
2. Vortex or mix by inversion for 10-15 seconds
c. Carbapenemase inderterminate: - Re-incubate all “No growth” plates up to 2 days (after
3. Add a 10-ug meropenem disk to each tube using sterile
i. Zone diameter of 16-18 mm reading all the plates, re-incubate)
forceps or add a single disk dispenser. Ensure the entire
ii. Zone diameter or ≥ 19 mm and the presence of - Perform subculture in BHI broth has sign of growth while
disk is immersed in the suspension
pinpoint colonies within the zone there are no growth on primary plates
4. Incubate at 35oC in ambient air for 4 hours ±15 minutes
iii. The presence or absence of a carbapenemase - If primary plates and BHI broth are positive, correlate the
5. Just before or immediately following completion of the
cannot be confirmed organisms isolated
TSB-meropenem disk suspension incubation, prepare a
0.5 McFarland suspension (using the colony suspension
method) of E. coli ATCC 25922 in nutrient broth or saline

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 Microbiology Section (Part 2)

1. Preliminary Report GROWTH ON PRIMARY SOLUTION. POSITIVE - If no colonies found in culture, report: “NO
- The preliminary report for the absence of growth FOR (IDENTIFIED ORGANISM) FROM GROWTH AFTER TWO (2) DAYS OF INCUBATION”
should be reported as: “NO GROWTH AFTER 24 THIOGLYCOLATE BROTH AFTER (DAYS OF 3. Record final report and date of release in the
HOURS OF INCUBATION”. INCUBATION)” (Susceptibility test results are designated logbook
included if applicable)
2. Final report - Final report with negative results for any REFERENCES
- Final report with presence of growth on both organism should be reported as: “NO GROWTH Notes from the discussion of Ms. Gracia M. Catulin, RMT
primary plates and BHI should be reported as: AFTER 4 DAYS OF INCUBATION” Notes from the synchronous session of Ms. Gracia M.
“POSITIVE FOR (IDENTIFIED ORGANISM)” Catulin, RMT
(Susceptibility test results are included if Record both preliminary and final report and date of release EACMC-Cavite video presentation for alternative medical
applicable) in the designated logbook technology internship program: Microbiology section
- Final report with presence of growth on BHI only
should be reported as “NO GROTH ON PRIMARY URINE
ISOLATION. POSITIVE FOR (IDENTIFIED 1. Preliminary report is not routinely done after 24 hours
ORGANISM) FROM BHI BROTH AFTER (DAYS OF of incubation for the presence and absence of growth
INCUBATION) 2. Three or >3 different colonies should be considered
- Final report with negative results for any and reported as “MIXED CULTURE” and suggests a
organism should be reported as: “NO GROWTH “REPEAT COLLECTION”
AFTER 4 DAYS OF INCUBATION” 3. Three or >3 different colonies should be considered if
the patient is an emergency case or in catheter.
Record both preliminary and final report and date of release Identify and perform susceptibility testing on
in the designated logbook predominant organisms and the colony count is
>100,000
3. Clinical significance 4. Final Report:
- If a typical pathogen is found from CSF culture it - Presence of growth: “(QUANTITY) CFU OF
is almost always significant. But many bacteria are (IDENTIFIED ORGANISM) PER ML OF URINE”
often significant but may occur as contaminants (Susceptibility test results are included if
in blood culture. Therefore, the finding maybe be applicable)
discussed with the clinician or place a note in the - Absence of growth: NO GROWTH AFTER 48
result form “Please correlate clinically” HOURS OF INCUBATION
5. Record final report and date of release in the
EXUDATES designated logbook
1. Preliminary Report
- The preliminary report for the absence of growth STOOL
should be reported as: “NO GROWTH AFTER 24 1. Preliminary report is not routinely done
HOURS OF INCUBATION”. 2. Final Report
- Final report with presence of growth on both
2. Final report primary plates and BHI should be reported as
- Final report with presence of growth on both “POSITIVE FOR (IDENTIFIED ORGANISM)”
primary plates and BHI should be reported as: - Stool specimen is known to have normal bacterial
“POSITIVE FOR (IDENTIFIED ORGANISM)” fecal flora. If no suspected pathogens observed or
(Susceptibility test results are included if tested, report: “NO IMPORTANT
applicable) ENTEROPATHOGEN ISOLATED”
- Final report with presence of growth on
thioglycolate only should be reported as: “NO

GAIL YVANA D. SARMIENTO | BSMT 4-3 10