International Clinical

Laboratories
Internship stay report
Departments of; Microbiology
Molecular biology
Core laboratories
Histopathology
Phlebotomy

By: Mihret genene

Date:

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Table of contents
1. Objective and introduction
2. Phlebotomy
3. Sample management
4. Core laboratories
5. Microbiology
6. Molecular biology
7. histopathology

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Abstract
The objective of this duration of practice is to develop the skills required in clinical
laboratory work. As a 3rd year medical laboratory technology student, I, have had
the chance to practice the science of clinical laboratory under supervision of senior
staff members, observing then performing procedures step by step as guided by
staff and standard operating procedures(SOP).

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Sample collection
Phlebotomy is the process of collecting appropriate samples from patients by
trained professionals or patients themselves depending on the sample required for
tests ordered. Samples include stool, blood, urine, sputum, swabbing’s, body fluids
etcetera.
BLOOD
Blood collection is performed by phlebotomists. it is performed in sterile manner
to protect the patients from environmental infection and also the phlebotomist from
needle stick injuries. Blood samples are collected in different collection tubes
according to the tests to be performed.
Methodology: vacutainer technique
1st draw; blood culture bottle
2nd draw; sodium citrate tubes
3rd draw; serum separator tubes(SST)
4th draw; heparin tube
5th draw; EDTA tube
URINE
This is collected by patients themselves provided with sterile screw cups. Care
must be taken so as to not contaminate urine samples with stool, menstrual blood,
other body fluids or environmental contaminants.
STOOL
Also collected by patients themselves, whilst avoiding body fluid of environmental
contaminants.
SPUTUM
Also collected by patients early morning after washing one’s mouth with sterile
saline and taking a few deep breaths into the lungs and collecting the thick mucus
like fluid that comes from deep within.

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BODY FLUIDS AND SWABBINGS
These are collected by trained physicians.

Sample management
In this department, samples are processed and grouped into their respective fate.
 Blood samples of SST and sodium citrate tubes are processed and then
centrifuged.
 Other samples are directly delivered to their respective laboratories.

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 Core laboratories
The core laboratory is a motherboard for various laboratory departments where
various specimens are tested using different test principles. It includes departments
of:
 Hematology and coagulation
 Immunology
 Clinical chemistry
 Serology
 Parasitology and
 Urinalysis
Hematology & coagulation assays
Hematology is the study of blood and blood disorders. In this department tests
concerning the cellular component of blood are performed. This mainly includes
complete blood counts (CBC), cd4+ lymphocyte counts, erythrocyte sedimentation
rate (ESR), prothrombin time & activated partial prothrombin time (PT & aPTT).
Complete blood count
A complete blood count, also known as a full blood count, is a set of medical
laboratory tests that provide information about the cells in a person's blood. The
CBC indicates the counts of white blood cells, red blood cells and platelets, the
concentration of hemoglobin, and the hematocrit.
Machinery: celldyn Ruby
Methodology: flow cytometry
Sample: EDTA whole blood
Parameters: RBC count, WBC count, PLT count, total hemoglobin concentration,
packed cell volume, neutrophil count & percentage, eosinophil count and
percentage, basophil count and percentage, monocyte count and percentage,
lymphocyte count and percentage, RBC indices (mean cell volume, mean cell
hemoglobin, mean corpuscular hemoglobin volume, red cell distribution width)
and mean platelet volume.
Reagents: WBC lyse, RBC lyse and sheath fluid

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Controls: sample of known value
Maintenance and background checks
Flags: WBC less than 2x109
RBC less than 2x1012
PLT less than 70x103
HGB less than 8g\dl
Coagulation
Coagulation, also known as clotting, is the process by which blood changes from a
liquid to a gel, forming a blood clot. It potentially results in hemostasis, the
cessation of blood loss from a damaged vessel, followed by repair. Coagulation
assays are performed to assess ability of an individual to heal after bleeding
injuries and progress of heparin therapy in patients with coagulation disorders.
Machinery: humaclot Duo plus
Methodology: photometry
Sample: sodium citrate anticoagulated plasma
Parameters: prothrombin time, international normalized ratio and partial
thromboplastin time.
Reagents: Tissue thromboplastin (PT)
Reagents 1&2 (aPTT)
Controls: level 1 & level 2
Flags: PT greater than 50
aPTT greater than 70
INR greater than 3
CD4+ lymphocyte count
A CD4 count is mostly used to check the health of your immune system if you are
infected with HIV (human immunodeficiency virus). HIV attacks and destroys
CD4 cells. Without treatment, HIV may destroy so many CD4 cells that your
immune system will have trouble fighting off infections.
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Machinery: Facs caliber
Methodology: flowcytometry
Sample: EDTA anticoagulated whole blood
Parameter: CD4+ count and %tage
CD8+ count and %tage
CD3+ count and %tage
CD45+ count and %tage
H\S percentage
Reagents: Lyse solution (external reagent)
Sheath fluid (internal reagent)

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 Urinalysis
A urinalysis is a test of your urine. It's used to detect and manage a wide range of
disorders, such as urinary tract infections, kidney disease and diabetes. A urinalysis
involves checking the appearance, concentration and content of urine. A complete
urinalysis consists of three components or examinations:
o physical (gross examination),
o chemical (dipstick), and
o microscopic.
Gross examination is done to determine visual consistency, elements, turbidity and
visual color of urine samples.
Consistency: clear
Turbid
Sediment
Color: Pale
Yellow
Brown
Red
Dipstick method and its indications:
Specific Gravity- cellular\organism presence
pH- acidity or alkalinity
Protein- cast and crystal presence
Leucocytes
Nitrites- bacterial presence
Blood- hemoglobinuria
Ketones-starvation
Glucose
Bilirubin

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 Urobilinogen
Microscopy; Urine microscopy is performed to examine for particles such as
human cells like RBC, WBC, epithelial cells, bacteria, casts and crystals etcetera.

Parasitology
Includes mainly microscopic stool examinations for protozoan or helminthic
parasites.
Machinery: light microscope
Methodology: wet mount preparation
Sample: fresh stool (examined within 30 minutes of specimen collection)
Parameters: presence of parasitic ova, cyst, trophozoite or etcetera
Reagent: Saline
Occult blood test to assess upper intestinal bleeding. By using gold coated
chromatography, hemoglobin in stool specimen resulting from lysed RBC
originating from upper intestinal bleeding is detected.
Machinery: -
Methodology: cassette chromatography
Sample: fresh stool
Parameters: Double colored line indicating a positive result and
Single colored line at the control region indicating a negative result
Reagent: saline for dilution of stool sample

Chemistry
Generally concerned with analysis of bodily fluids, mainly serum and plasma, for
diagnostic and therapeutic purposes. It makes use of simple chemical reaction tests
for various components of blood and urine for measurement of enzyme activities,
proteins, lipid, electrolytes, minerals and so on by methods of photometry,
potentiometry and spectrophotometry.

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Machinery: alinity c
Method: Photometric, Potentiometric
Sample: Serum, plasma, urine, cerebrospinal fluid, hemolysate, whole blood
Parameters:
Electrolytes Miscellaneous Liver function tests
o Sodium o Glucose o Total protein (serum)
o Potassium o C-reactive protein o Bilirubin; direct;
o Chloride o Glycated hemoglobin indirect; total
o Bicarbonate (HbA1c) o Aspartate
o Uric acid transaminase (AST)
o Arterial blood gases o Alanine transaminase
([H+], PCO2, PO2) (ALT)
o Adrenocorticotropic o Gamma-glutamyl
hormone (ACTH) transpeptidase (GGT)
o Alkaline phosphatase
(ALP)
Cardiac markers Minerals Renal (kidney) function
o Troponin o Calcium tests
o Myoglobin o Magnesium o Creatinine
o Phosphate o Blood urea nitrogen
o Potassium (BUN)
Blood disorders
o Iron
o Transferrin
o TIBC
o Vitamin B12
o Vitamin D
o Folic acid

Reagents: up to 70 different types

Immunology
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The specificity of the bond between antibody and antigen has made an excellent
tool for the detection of substances by a variety of diagnostic techniques.
Antibodies specific for a desired antigen (and vice versa) can be conjugated with
an isotopic (radio) or fluorescent label or with a color-forming enzyme in order to
detect it.
Machinery: alinity i
Method: Chemiluminescence
Sample: Serum, plasma, whole blood, urine
Parameters:
CANCER ASSAYS CARDIAC INFECTIOUS DISEASE
AFP ASSAYS ASSAYS
Beta2 Microglobulin BNP HEPATITIS
CA 125 II Galectin-3 Anti-HAV IgG
CA 15-3 Troponin-I (High Anti-HAV IgM
CA 19-9 (CA 19-9 XR) Sensitive) Anti-HBc IgM
CEA Anti-HBe
CYFRA 21-1 Anti-HBs
HE-4 Anti-HCV
Immunoglobulin A (IgA) HBeAg
Immunoglobulin E (IgE) HBsAg (Quantitative)
Immunoglobulin G (IgG) HCV Ag
Immunoglobulin M OTHER INFECTIOUS
(IgM) DISEASE
Kappa Light Chain Chagas
Lambda Light Chain EBV EBNA-1-IgG
Pepsinogen I EBV VCA IgG
Pepsinogen II EBV VCA IgM
PIVKA-II Syphilis TP
ProGRP RETROVIRUS
PSA, Free Anti-HTLV-I/HTLV-II
PSA, Total TORCH
SCC CMV IgG
CMV IgG Avidity
CMV IgM
HSV-1 IgG
HSV-2 IgG
Rubella IgG
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 Rubella IgM
Toxo IgG
Toxo IgG Avidity
Toxo IgM

Reagents: up to 47

Serology
Serology is the scientific study of serum and other body fluids. In practice, the
term usually refers to the diagnostic identification of antibodies in the serum. Such
antibodies are typically formed in response to an infection (against a given
microorganism), against other foreign proteins (in response, for example, to a
mismatched blood transfusion), or to one's own proteins (in instances of
autoimmune disease). In either case, the procedure is simple.
Several methods can be used to detect antibodies and antigens, including ELISA,
agglutination, precipitation, complement-fixation, and fluorescent antibodies and
more recently Chemiluminescence.
The main serological tests performed in the ICL core laboratory are
o TPHA
o RPR and
o HPYag
Treponema pallidium haemagglutination test (TPHA)
Is a standard treponemal test used to confirm a positive non-treponemal test. The
principle is that; patient diluted serum is mixed in a well of microtitration plate
with sheep or avian RBCs coated (sensitized) with T.P Ag (Nichols' strain). If Ab
is present in the serum, the sensitized RBCs are aggregated and they settle in a
characteristic pattern in the bottom of the well. Un agglutinated cells form a button
or smooth ring at the bottom of the well.

Method:

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 o Add 190 μl of the diluent to one well.
o Add 10 μl of specimen or Positive Control or Negative Control
to the same well and mix thoroughly.
o Transfer 25 μl of diluted control or diluted specimen from dilution
step to test well.
o Transfer 25 μl of diluted control or diluted specimen dilution step to
control well.
o Re-suspend the Test Cells and the Control Cells by shaking
the vial and examine for complete suspension.
o Add 75 μl of Test Cells to test well and 75 μl of Control Cells
to the control well.
o Final specimen dilution is 1: 80.
o Mix wells thoroughly.
o Incubate at room temperature (15-30 °C) on a vibration-free surface, in a
dark room for a minimum of 45 minutes.
o Read the settling patterns. Agglutination patterns are stable for at
least three hours if undisturbed.
Rapid plasma reagenic test /RPR/
A non treponemal flocculation test used to detect regain Abs in serum and plasma
from syphilitic patient. The Ag is a modified VDRL Ag containing
micropatrticulate carbon /charcoal which enhances the visual difference between
positive and negative result. Ag is composed of VDRL Ag (which has added
EDTA, Choline chloride, Merthiolate and particles of carbon).
Specimen: serum or plasma
Turbid hemolyzed or contaminated samples should not be used. Storage for several
days at 2-8º C or -20⁰ C is possible.
Machinery: shaker\mixer
Method:
 0.05ml of serum or plasma spread throughout the circle.
 1/60 ml of well mixed Ag suspension.
 Rotate manually or automatic shaker for 8’
 Immediately after rotation read results macroscopically in good
light
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► Positive (Reactive) =large aggregate in center or periphery of the test circle.

► Weak reactive =small aggregate around the edge of the test circle.

► Negative (Non-Reactive) =no visible aggregate

Helicobacter pylori blood test

A rapid test for the qualitative detection of antibodies to Helicobacter pylori (H. pylori) in whole blood,
serum, or plasma. In this test procedure, anti-human IgG is immobilized in the test line region of the
test. After specimen is added to the specimen well of the device, it reacts with H. pylori antigen coated
particles in the test. This mixture migrates chromatographically along the length of the test and interacts
with the immobilized anti-human IgG. If the specimen contains H. pylori antibodies, a colored line will
appear in the test line region indicating a positive result. If the specimen does not contain H. pylori
antibodies, a colored line will not appear in this region indicating a negative result. To serve as a
procedural control, a colored line will always appear in the control line region, indicating that proper
volume of specimen has been added and membrane wicking has occurred.

Method:

o Remove test device from pouch and label is with sample ID

o Using a Pasteur pipette provided with the kit make few drops of test sample on to strip end
indicated by arrows
o Wait 5-10 minutes to interpret results.

POSITIVE: Two lines appear. One colored line should be in the control line region and another

apparent colored line should be in the test line region.

NEGATIVE: One colored line appears in the control line region. No line appears in the test line

region.

INVALID: Control line fails to appear. Insufficient specimen volume or incorrect procedural

techniques are the most likely reasons for control line failure.

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 Microbiology
Diagnostic microbiology is the study of microbial identification. Methods used in
diagnostic microbiology are often used to take advantage of a particular
difference in organisms and attain information about what species it can be
identified as, which is often through a reference of previous studies.
The main responsibilities of diagnostic microbiology laboratories are
o Microbial identification and
o Antimicrobial sensitivity testing

Microbial identification
Microbiology laboratories perform identification based on several procedural
steps through culturing and biochemical tests. A microbiological culture, or
microbial culture, is a method of multiplying microbial organisms by letting them
reproduce in predetermined culture medium under controlled laboratory
conditions.
Culture media is a gel or liquid that contains nutrients and is used to grow
bacteria or microorganisms. They are also termed growth media. Different cell
types are grown in various types of medium.
1. Mackonkey agar: is a selective and differential media for gram negative
bacteria. It differentiates lactose fermenters from non-lactose fermenters
which is demonstrated by pink colonies for NLF and yellow colonies for LF
bacteria.
2. Blood agar: is an enriched media made from basic media by adding 5
percent sheep blood. It allows growth of fastidious aerobic bacteria.
3. Chocolate agar: also an enriched media for growth of fastidious anaerobic
bacteria like Neisseria spp.
4. XLD agar: a selective media for salmonella and shigella.
5. Muller Hinton agar: used for antimicrobial sensitivity testing.
6. MIGT: a liquid media intended for the detection and recovery of
mycobacteria.

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 7. Lowenstien-jensen agar: an egg based solid media intended for the
detection and recovery of mycobacteria.
Biochemical tests are used to differentiate different organisms based on their
genus and species characteristics. They are performed on pure culture.
1. Catalase test: differentiates catalase producing staphylococci from non-
catalase producing streptococci
2. Coagulase Test: differentiates coagulase producing s. aureus from other
staph spp
3. Urease test: for urease enzyme activity testing. It is important in
differentiating enterobacteriae like proteus and y. enterocolitica
4. Indole test: detects the ability of an organism to produce indole from
Tryptophan present in the medium which is important in
enterobactericae identification.
5. Citrate utilization test: detects the ability of an organism to use citrate
as its only source of carbon for identification of enteric bacteria.
6. Lysine decarboxylase test (LDC): to differentiate members of
Enterobacteriaceae on the basis of their ability to produce the enzyme
decarboxylase.
7. Triple sugar Iron (TSI) & Hydrogen sulfide production (H2S): looks for
fermentation of glucose, lactose, and sucrose and checks if hydrogen
sulfide (H2S) and gas is produced in the process.
8. Kligler Iron Agar (KIA): same as TSI.
9. Motility-Indole-Urea (MIU): a test that checks for bacterial motility, urea
and indole production all at once.
10.Fermentation of carbohydrates

Bactec phoenix BD
Is an automated microbiology system for direct identification and susceptibility
testing of pure bacterial cultures. ID based on colorimetric and fluorescence
readings without the need for supplemental tests or reagent addition. AST is
based on a dual indicator system (redox and turbidity readings) for optimal
performance.
Machinery: Bactec phoenix BD

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Methodology: colorimetry and fluorescence
Sample: pure culture colonies from urine, wound swab, throat swab, CSF and
other specimens.
Parameters: identification and AST
Reagents: provided with test panel kit

Mycobacteria tuberculosis culture

Mycobacterial culture is a lengthy and complex procedure which is prone with
many technical errors and environmental contamination. Thus it is performed in a
containment laboratory sealed and separated from the rest of the workspace.
Machinery: BACTEC MGIT 960
Methodology: fluorescence of an oxygen sensor
Sample: sputum
Parameters: mycobacterial growth and drug sensitivity testing for streptomycin,
isoniazid, ethambuthol and rifampicin.
Reagents: MGIT liquid media, sodium hydroxide for sample decontamination,
phosphate buffered saline for washing.
The BACTEC MGIT 960 instrument is a fully automated system that exploits the
fluorescence of an oxygen sensor to detect growth of mycobacteria in culture.
The incubation process takes about 3-8 weeks and thus making is very time
consuming aside from the fact that it is laborious work.

Interferon gamma release assay (IGRA)

IGRA is a screening test for exposure to M. TB in individuals especially young
children who are unable to obtain sputum samples and asymptomatic individuals.
Machinery: quantiFERON-TB Gold Plus ELISA
Methodology: ELISA
Sample: heparin anticoagulated whole blood

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Parameters: qualitative M. TB screening (positive or negative)
Reagents: provided with the test kit
The QFT-Plus assay uses specialized blood collection tubes, which are used to
collect whole blood. Incubation of the blood occurs in the tubes for 16 to 24
hours, after which, plasma is harvested and tested for the presence of IFN-γ
produced in response to the peptide antigens. The QFT-Plus test is performed in
two stages. First, whole blood is collected into each of the QFT-Plus Blood
Collection Tubes, which include a Nil tube, TB1 tube, TB2 tube, and a Mitogen
tube. Alternatively, blood may be collected in a single generic blood collection
tube that contains lithium heparin as the anticoagulant, and then transferred to
QFT-Plus tubes.
The Mitogen tube is used with the QFT-Plus test as a positive control. This may be
important where there is doubt as to the individual’s immune status. The Mitogen
tube also serves as a control for correct blood handling and incubation. The QFT-
Plus tubes should be incubated at 37°C as soon as possible, and within 16 hours of
collection. Following a 16 to 24-hour incubation period, the tubes are centrifuged,
the plasma is removed and the amount of IFN-γ (IU/ml) is measured by ELISA.
A QFT-Plus assay is considered positive for an IFN-γ response to either TB Antigen
tube that is significantly above the Nil IFN-γ IU/ml value. The plasma sample from
the Mitogen tube serves as an IFN-γ positive control for each specimen tested. A
low response to Mitogen (<0.5 IU/ml) indicates an indeterminate result when a
blood sample also has a negative response to the TB antigens. This pattern may
occur with insufficient lymphocytes, reduced lymphocyte activity due to improper
specimen handling, incorrect filling/mixing of the Mitogen tube, or inability of the
patient’s lymphocytes to generate IFN-γ. The Nil tube adjusts for background
(e.g., excessive levels of circulating IFN-γ or presence of heterophile antibodies).
The IFN-γ level of the Nil tube is subtracted from the IFN-γ level for the TB Antigen
tubes and Mitogen tube.

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 Molecular biology
Molecular diagnostics, also called molecular pathology, involves taking DNA or
RNA, the unique genetic code found in our cells, and analyzing the sequences for
red flags that can pinpoint the potential emergence of a specific disease. These
technologies generally can be grouped into three approaches: polymerase chain
reaction (PCR), hybridization, and next-generation sequencing (NGS).

Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is a technology used for quick and easy
amplifying DNA sequences, which is based on the principle of enzymatic
replication of the nucleic acids. This method has in the field of molecular biology
an irreplaceable role and constitutes one of the basic methods for DNA analysis.
Zybio SARS-CoV-2 Nucleic Acid Detection Kit
Real time PCR
Qualitatively detects the RNA of SARS-CoV-2 in the specimen through measuring
the change of fluorescence signal intensity during RT-PCR amplification with
specific primers and probes against the conserved region of ORF 1ab and N gene,
using One Step RT-PCR method
The extraction kit absorbs the nucleic acid of the virus by magnetic beads, only
takes 4 minutes for cell breaking and absorption of nucleic acid. Only one washing
is required during the extraction to get highly purified nucleic acid. Manual
extraction requires magnetic separator, centrifuge, dry bath, etc.
Machinery: Nucleic Acid Extraction Kit
Nucleic Acid Isolation System EXM3000
SARS-CoV-2 Nucleic Acid Detection Kit
Fluorescence PCR analyzer
Methodology: PCR
Sample type: nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid, stool,
etc.

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Sample volume: 10ul
Reagents: extraction reagent, elution buffer, magnetic beads solution, proteinase
k, and instrument system solution.

ID Now
Is a rapid, instrument-based, isothermal system for the qualitative detection of
infectious diseases. Our unique ID NOW™ isothermal nucleic acid amplification
technology provides molecular results in just minutes.
ID NOW COVID-19 is a rapid (13 minutes or less), instrument-based isothermal
test for the qualitative detection and diagnosis of SARS-CoV-2 from nasal,
nasopharyngeal and throat swabs. The ID NOW COVID-19 kit contains all
components required to carry out an assay for SARS-CoV-2 on the ID NOW
Instrument.
To perform the assay, the Sample Receiver and Test Base are inserted into the ID
NOW Instrument. The sample is added to the Sample Receiver and transferred via
the Transfer Cartridge to the Test Base, initiating target amplification. Heating,
mixing and detection are provided by the instrument.

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 Histopathology
Histopathology is the diagnosis and study of diseases of the tissues, and involves
examining tissues and/or cells under a microscope. Histopathologists are
responsible for making tissue diagnoses and helping clinicians manage a patient's
care.
ICL’s histopathology laboratory services are mainly divided into two
o Biopsy and
o Fine needle aspirations (FNA)

Biopsy is a procedure to remove a piece of tissue or a sample of cells from your

body so that it can be tested in a laboratory. Pathological examination of a biopsy
sample has various procedures such as fixation and processing, embedding and
microtomy, deparaffinization and staining, and finally coverslipping.
Machinery: microscopy, microtome
Methodology: sectioning and staining
Sample: surgically removed tissue biopsy
Parameters: presence of fungal, bacterial or viral pathogens and cancerous cell
morphology
Reagents: formalin, hematoxylin and eosin stain, alcohol, xylene, isopropanol and
paraffin for embedding.
The tissue processing procedure includes;
 Obtaining a fresh specimen.
 Fixation in a liquid fixing agent (fixative) such as formaldehyde solution
(formalin). This will slowly penetrate the tissue causing chemical and
physical changes that will harden and preserve the tissue and protect it
against subsequent processing steps.
 Dehydration Because melted paraffin wax is hydrophobic (immiscible with
water), most of the water in a specimen must be removed before it can be
infiltrated with wax. This process is commonly carried out by immersing

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 specimens in a series of ethanol (alcohol) solutions of increasing
concentration until pure, water-free alcohol is reached.
 Clearing by use of an intermediate solvent that is fully miscible with both
ethanol and paraffin wax. This solvent will displace the ethanol in the
tissue, then this, in turn, will be displaced by molten paraffin wax.
 Wax infiltration wax is liquid at 60°C and can be infiltrated into tissue at this
temperature then allowed to cool to 20°C, where it solidifies to a
consistency that allows sections to be consistently cut.
 Embedding or blocking out by forming into a “block” which can be clamped
into a microtome for section cutting. This step is carried out using an
“embedding center” where a mold is filled with molten wax and the
specimen placed into it. The specimen is very carefully orientated in the
mold because its placement will determine the “plane of section”.

FNA is a diagnostic procedure used to investigate lumps or masses. In this

technique, a thin, hollow needle is inserted into the mass for sampling of cells
that, after being stained, are examined under a microscope.
Machinery: microscopy
Methodology: staining
Sample: fine needle aspirate from lumps
Parameters: cytology
Reagents: Giemsa staining
Method:
o Samples are obtained by inserting a needle into the lump area and
aspirating as much fluid as possible
o The sample is placed onto a microscopic slide and a film is made by running
another microscopic slide over the specimen
o Slides are allowed to dry
o Fixation
o Staining with giemsa stain
o Observing under a microscope for any abnormal cellular morphology or
foreign invasion
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