Laboratory | Ms. Dalida, F.

| Midterms

Activity 1 – Handwashing 2. Apply soap, preferably 9. Make thumb holes at the border of
Activity 2 – Donning and Doffing of PPE antimicrobial. the cuff and sleeves.
Activity 3 – N/A 3. Rub to form a lather, create 10. Put on N95 Respirator.
Activity 4 – OPS and NPS Collection friction, and loosen debris. 11. Perform seal check.
Activity 5 – Micropipetting Thoroughly clean between the 12. Put on hood.
fingers and under the fingernails 13. Put on outer apron.
for at least 20 seconds; include 14. Put on outer gloves.
Activity 1 – Handwashing thumbs and wrists in the cleaning. 15. Put on face shield or goggles.
4. Rinse hands in a downward 16. Let the trained observer check the
Handwashing position to prevent worn PPE.
- The most important procedure in recontamination of hands and
isolation technique. wrists. DOFFING
- First and last step of all 5. Obtain paper towel from the 1. Inspect PPE for visible contamination
procedures. dispenser. or tears.
- Does not sterilize hands, but 6. Dry hands with paper towel. 2. Disinfect outer gloves.
removes surface contaminants, 7. Turn off faucets with a clean 3. Remove apron.
dead skin, and surface organisms. paper towel to prevent 4. Inspect the PPE again.
1. Create an infographic of proper contamination. 5. Disinfect outer gloves.
handwashing. 6. Remove the boot covers.
2. What is the function of Infection 7. Disinfect outer gloves.
Control Department in the Activity 2 – Donning and Doffing of PPE 8. Remove outer gloves.
hospital? 9. Inspect and disinfect inner gloves.
3. When is handwashing performed? DONNING 10. Remove goggles.
1. Ensure that a Trained Observer is 11. Disinfect inner gloves.
Handwashing Equipment: available to verify compliance with 12. Remove hood.
- Antimicrobial soap all steps. 13. Disinfect inner gloves.
- Paper towels 2. Ensure that all the needed supplies 14. Remove coverall.
- Running water are available. 15. Disinfect inner gloves.
- Waste container 3. Prepare for donning of PPE. 16. Change inner gloves.
4. Inspect PPE Items prior to putting on. 17. Perform hand hygiene.
Handwashing Procedure: 5. Put on boot covers. 18. Put on a new pair of gloves.
1. Wet hands with warm water. Do 6. Perform hand hygiene. 19. Remove N95 Respirator.
not allow parts of body to touch 7. Put on inner gloves. 20. Disinfect gloves.
the sink. 8. Put on cover-all/gown. 21. Disinfect washable boots.

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22. Disinfect and remove gloves. o Nursery Precautions

23. Perform hand hygiene. Standard Precaution Guidelines o Healthy Lifestyle
24. Inspect for any contamination. ❖ Handwashing
❖ Gloves • Infectious Agent
(Ref: Department of Health) ❖ Laboratory gown - Bacteria
❖ Mask/Eye Protection/Face shields - Fungi
❖ Occupational Health and - Parasites
Laboratory Safety Bloodborne Pathogens - Viruses

➢ Universal Precautions • Reservoir

- Provided guidelines that Hazards - Humans
recommend precautions in - Animals
handling body fluids and human Biological Hazard - Insects
tissues for all patients regardless of • Microorganisms present in clinical - Fomites
their blood-borne infection status. samples that can potentially cause - Blood/Body Fluids
➢ Body Substance Isolation an infection or disease. - Break the link:
- Not limited to bloodborne • Infection Control – Procedures to o Disinfection
pathogens; they consider all body control and monitor infections o Hand Hygiene
fluids and moist body substances occurring within their facilities
to be potentially infectious. • Chain of Infection - a continuous link • Portal of Exit
Handwashing/ sanitizing is among six elements: infectious agent, - Nose
recommended after removal of a reservoir, a portal of exit, a means - Mouth
gloves of transmission, a portal of entry, and - Mucous membranes
➢ Standard Precaution a susceptible host - Specimen Collection
- Combined the major features of - Break the link:
UP and BSI guidelines • Susceptible Host o Sealed biohazardous
- Patients o Sealed specimen
✓ CDC – Centers for Disease Control - Elderly containers
and Prevention - Newborn o Hand hygiene
✓ OSHA – Occupational Safety and - Immunocompromised o Standard precautions
Health Administration - Health-care workers
✓ HICPAC – Healthcare Infection - Break the link: • Means of transmission
Control Practices Advisory o Immunizations - Droplet
Committee o Patient Isolation - Airborne

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- Contact • Biosafety Cabinet - Containment severity. Example: Staphylococcus

- Vector barriers that protects the workers from aureus.
- Vehicle the aerosolized transmission of - LABORATORY PRACTICES
- Break the link organisms o Access to the laboratory is
o Hand hygiene - Class I – Uses an exhaust fan to restricted when work is
o Standard Precautions move air inward through the open being conducted.
o PPE front - SAFETY EQUIPMENT
o Patient isolation - Class II – Air is pulled inward and o Personal Protective
downward by a blower Equipment
• Portal of entry - Class III – Has a self-contained o BSC
- Nose ventilated system o Autoclave
- Mouth - FACILITY CONSTRUCTION
- Mucous Membrane • Biosafety Levels o The laboratory has self-
- Skin • Biosafety Level 1 - Microbes are not closing doors.
- Unsterile equipment known to consistently cause disease o A sink and eyewash are
- Break the link: in healthy adults. Example: readily available.
o Hand hygiene nonpathogenic strain of E. coli. o Door
o Standard precautions - LABORATORY PRACTICES • Biosafety Level 3 - Microbes can be
o PPE o Standard lab practices either indigenous or exotic, and they
o Sterile equipment o Work can be performed in can cause serious or potentially lethal
open benches disease through respiratory
• Disposal of Biological wastes - SAFETY EQUIPMENT transmission. Respiratory transmission
- Sterilization: Destruction of all o Personal Protective is the inhalation route of exposure.
forms of life including bacterial Equipment Example: Mycobacterium
spores - FACILITY CONSTRUCTION tuberculosis.
- Disinfection: Refers to a process of o Sink availability - LABORATORY PRACTICES
eliminating a definite scope of of o Working space separated o Medical surveillance and
microorganisms working space from the immunization
- AUTOCLAVE 121*C, 15 mins, 15 psi facility by a door o Access to the laboratory is
- DRY HEAT STERILIZATION • Biosafety Level 2 - Microbes pose restricted and controlled at
- FILTRATION HEPA Filter - High Efficiency moderate hazards to laboratorians all times
- RADIATION Particulate Air Filter and the environment. The microbes - SAFETY EQUIPMENT
- CHEMICALS are typically indigenous and o Appropriate PPE and
associated with diseases of varying respirators
Sodium Hypochlorite
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o BSC Chemical Hazard

- FACILITY CONSTRUCTION
o Have sustained directional
airflow
o two sets of self-closing and
locking doors
-
• Biosafety Level 4 - Microbes in a BSL-4
MUST BE DISPLAYED ON THE
lab are dangerous and exotic, posing • Chemical Hygiene Plan DOORS OF ALL AREAS WHERE
a high risk of aerosoltransmitted - Appropriate work practices RADIOACTIVE MATERIAL IS
infections. Infections caused by these - SOP
microbes are frequently fatal and PRESENT
- PPE
without treatment or vaccines. - Engineering controls
Example: Ebola and Marbug virus. - Employee training
- LABORATORY PRACTICES - Medical consultation guidelines
o Change clothing before • Material Safety Data Sheet
entering. - Physical and chemical
o Shower upon exiting. characteristics -
o Decontaminate all - Fire and explosion potential MONITORING OF ELECTRICAL
materials before exiting. - Reactivity Potential WIRINGS AND EQUIPMENTS. ALL
- SAFETY EQUIPMENT - Health hazards and emergency EQUIPMENTS MUST BE GROUNDED
o Appropriate PPE first aid WITH THREEPRONGED PLUGS
o Positive pressure, air- - Methods for safe Handling and
supplied full body suit disposal
o BSC Class III Fire Hazard
- FACILITY CONSTRUCTION
o Separate facility Other Hazards
o Dedicated supply and
exhaust of air

Joint Commission on Accreditation of

Healthcare Organizations
-
PUNCTURE PROOF CONTAINERS
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• RACE – Rescue, Alarm, Contain, SPECIMEN COLLECTION OF

Evacuate/Extinguish NASOPHARYNGEAL SWAB AND
• PASS – Pull the pin; Aim at the base of OROPHARYNGEAL SWAB
the Fire; Squeeze Handles; Sweep
Nozzles Side to Side SUPPLIES/EQUIPMENT NEEDED
✓ COVID-19 Case Investigation Form
(CIF) and RITM linelist
Activity 4 – OPS and NPS Collection ✓ Virus Transport Media (VTM) or
Universal Transport Media (UTM) or
Severe Acute Respiratory Syndrome – Sample Storage Reagent (Sansure)
Corona Virus - 2 (SARS – CoV – 2) ✓ Nasopharyngeal swab, Sterile
- COVID 19 Dacron/Rayon swab with pliable
- Wuhan, China shaft
- December 2019 ✓ Oropharyngeal swab, Sterile
- Respiratory Symptoms: cough, Dacron/Rayon swab with plastic shaft
shortness of breath, or difficulty of ✓ Sterile tongue depressor
breathing ✓ Test Tube rack
- Less common symptom: diarrhea ✓ Resealable plastic bags (zip lock)
- Culture specimens: blood, urine, ✓ Laboratory sealing film (parafilm)
feces, swabs, and tissues ✓ Masking tape
- Routine Diagnosis: ✓ Permanent tube marker
Nasopharyngeal and ✓ Scissors
Oropharyngeal swabs for RT-PCR ✓ PPE (Laboratory gown, gloves,
testing N95mask, goggles)
o RT-PCR testing is the gold ✓ Refrigerator
standard and the
confirmatory testing for ✓ COLLECTION KIT
SARS – CoV – 2. o Virus Transport Medium
o Nasopharyngeal swab
o Oropharyngeal swab
✓ COPAN SAMPLE COLLECTION KIT
o Universal Transport Medium
(UTM) or equivalent

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o Copan Flocked Swab or GUIDELINES PRIOR TO SPECIMEN

equivalent COLLECTION
o Sample Storage Reagent
(Sansure) and OPS INFECTION CONTROL GUIDELINES
✓ Personal protective equipment: wear
DIRECTIONS FOR USE OF SWAB N95 mask and disposable gloves.
➢ Swabs should not be used if ✓ When completed, dispose of all PPE
o there is evidence of damage and other contaminated materials in
or contamination the appropriate trash bin.
o the expiration date has passed RELEVANT ANATOMY: THE RESPIRATORY ✓ Wash hands thoroughly with soap
o the swab package is SYSTEM and water or alcohol-based hand gel
damaged before AND after the procedure
o there are other signs of
deterioration SPECIMEN COLLECTION POLICIES
➢ During sampling, the swab shall only ✓ Specimens shall be collected within
come in contact with the suspected 10 days from onset of illness.
infection. ✓ Only qualified and trained staff shall
➢ Do not use excessive force when perform the procedures.
collecting swab samples from ✓ Do NOT use wooden and cotton
patients as this may result in swabs.
accidental breakage of the swab ✓ Check for integrity of the OPS/NPS
shaft. swabs and do not use beyond
➢ Swabs are for single use only. expiration.
✓ Use only the approved kits for
TECHNICAL NOTES: CORRECT HANDLING specimen collection.
OF SWAB ✓ Correctly identify the patient and
label the UTM tube prior to collection.
✓ Label the tube with the patient’s full
name, age and date of collection.
The information on the label must
MATCH the information on the CIF.
✓ Remove possible visual obstructions.

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✓ Strictly follow infection control refrigerator, use a thermo bag/box with SPECIMEN COLLECTION POLICIES
guidelines prior to each procedure. 4-6 icepacks
(Protect yourself from the patient) Sample
Virus
SPECIMEN COLLECTION PROPER Storage
Transport
SPECIMEN COLLECTION PROPER OROPHARYNGEAL SWAB (OPS) Reagent
Medium
NASOPHARYNGEAL SWAB (NPS) Step 1: Have the patient seated (Sansure)
Step 1: Using the swab, visually measure comfortably. Have the patient open his No Spx RT or 2-8 C - 20 C
from the base of the nostril towards the mouth. With gloved hands, hold down
w/ OP/NPS 2-8 C 2-8 C
auditory pit. Divide the length into half in the tongue with a sterile tongue
order to know into what extent will be depressor Have the patient say “AAH͟” to
inserted into the nostril (usually 5–6 cm in elevate the uvula - Do not use UTM/VTM if there is
adults) to ensure that it reaches the Step 2: Use a sweeping motion to swab evidence of contamination
posterior pharynx. Alternatively, you may the posterior pharyngeal wall and - Do not use expired UTM/VTM
use a ruler for more accurate tonsillar fossa. Avoid swabbing the soft
measurements palate. Do not touch the tongue with the
Step 2: With the patient seated, tilt the swab tip Activity 5 – Micropipetting
head slightly backwards. Insert the swab Step 4: Place the OPS immediately in the
into the nostril parallel to the palate same UTM with NPS. Cut the end of the Pipetting is an essential skill student need
Step 3: Insert the swab into the nasal shaft that sticks out of the tube and close to master to successfully execute
cavity until a slight resistance is met. the tube tightly. Secure the cap with laboratory activities in many fields of
Rotate the swab and apply a little force Parafilm to prevent leakage during science, including molecular biology.
to take large quantities of mucosa. transport. Molecular biology laboratories utilize
Repeat in the other nostril using same micropipetting instruments
swab Best Practices (micropipettors) as tools to accurately
Step 4: Place the NPS immediately in the Ask patient to remove extra secretions transfer very small volumes (microliters) of
UTM tube to avoid drying of the swab. before procedure. liquid from one container to another.
Break/Cut the end of the shaft that sticks ✓ Ask patient to close his or her eyes. Skillful use of a micropipettor is a critical
out of the tube (break point) and close ✓ Visually estimate the depth of component of molecular biology
the tube tightly. Secure the cap with insertion. laboratory procedures. To perform this
Parafilm to prevent leakage during ✓ Label the tube first PRIOR to skill effectively requires practice. Often,
transport collection. failure to perform an accurate
Step 5: Transport the specimen to the ✓ REMEMBER: Your safety first and micropipetting step in the laboratory
laboratory and immediately store inside foremost. procedure will result in poor results or no
refrigerator (2- 8C). • If site is far from a results at all. In this activity, use and care
of a micropipettor is described and
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present activities designed to help 5. Never lay the micropipettor on its side 5. Allow the plunger to slowly return to
conceptualize working with small when the pipette tip contains liquid. the UP position. Remember; do not let
volumes and develop skill using this 6. To obtain the most accurate it "snap" to the up position. Then
important tool for molecular diagnostics. measured volume, use the carefully withdraw the tip from the
micropipettor best suited for your sample making sure there are no air
Micropipettors can dispense various pipetting needs. bubbles.
volumes. Generally, the plunger button 7. Avoid dropping and rough handling 6. To dispense the liquid, gently touch
indicates the maximum volume of the micropipettor. the tip to the side of the receiving
(microliters) that the pipette is designed 8. If liquid has gone up into the barrel of vessel. Press the plunger past the first
to handle. Micropipettors came in the micropipettor, inform one of the stop to the second stop. With the
various sizes ranging from 5ul to 1000 ul instructors. plunger fully pressed, withdraw the tip
capacity. The volume indicator is read 9. See the manufacturer's instruction carefully, wiping residual drops
from top to bottom (Figure 2B). For P-2, P- booklet for more specific information against the vessel wall.
10, P-20, P100, and P-200, black digits regarding the use and care of the 7. Allow the plunger to slowly return to
indicate microliters and red digits micropipettor. the UP position.
indicate tenths and hundredths of 8. Discard the tip by depressing the tip
microliters. For P-1000, red digits indicate Instructions for pipetting liquids using a ejector button.
milliliters and black digits indicate micropipette.
microliters. Not all micropipettors have 1. Hold the micropipettor in one hand.
colored digits on their volume indicators. With the other hand, turn the volume
adjustment knob to the desired
Maintenance and care of the setting.
micropipettor: Important things to 2. Attach a new disposable tip to the
remember regarding the use and care of pipette shaft. Be sure the tip is
the micropipettor. properly attached and has a good
1. Apply uniform pressure to the seal.
plunger. 3. Press the plunger to the first stop
2. Release the plunger slowly and with where you feel a slight resistance. This
smoothness. represents the volume displayed on
3. Do not immerse the pipette tip the digital indicator.
completely into a solution. 4. Holding the micropipettor vertically,
4. Maintain a minimal angle when immerse the tip a few millimeters into
pipetting. the sample while holding the plunger
at the first stop.

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Micropipetting Basics Based on Volume Infectious

- Fixed Volume Micropipette Biological Bacteria, viruses,
Accuracy vs Precision o Viscous Suspension patient and
- Accuracy: how close a - Adjustable Volume Micropipette (Hazardous) environmental
measurement is to the true value samples
- Precision: the reproducibility of Based on Pipetting Mechanism DNA extraction
the measurement – how close the - Mechanical/Manual Reaction (DNA +
measurements to each other o pressure/with spring Buffer + ProK
- Ensures the experiments are both - Electronic Enzyme)
successful and reproducible o Electronic Button
DNA
Parts of a Micropipette Uses of Micropipette Amplification
- Volume Adjustment Knob ➢ Clinical and microbiological Reaction (DNA +
- Tip Ejector Button laboratory Buffer + Taq
Reactions
- Ejector Arm ➢ Chemical Laboratory Enzyme +
- Digital Volume Display Window ➢ Forensic Laboratory Nucleotides)
- Plastic Shaft ➢ Pharmaceutical Laboratory
- Tip Cone ➢ Food and beverage industry Bacterial
- Pipette Tip Transformation
Common (Nutrient Media +
Examples
Based on Principle: Reagents Bacteria Cells +
- Air Displacement Micropipette TE Buffer, Water, DNA + Calcium
o Temperature and viscosity stop solutions, Chloride)
Aqueous Soln.
- Positive Displacement electrophoresis
Micropipette buffer, PBS buffer The ins and outs of Pipetting
o Piston is directly in contact Glycerol, oil, ❖ Today, most labs are equipped with
with the liquid Viscous and
ethanol, piston displacement micropipettes
o No temperature and Volatile Soln.
formaldehyde ❖ Inside these pipettes are solid disk
viscosity Acids, bases, and tightly fitted tube which moves
Corrosive and
radioactive when a plunger is depressed
Based Number of Channels Hazardous Soln.
isotopes o Fixed Volume
- Single Channel Biological DNA, RNA, o Adjustable Volume
- Multichannel Suspension (Non- Restriction
Hazardous) Enzymes
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Metric Volumes (Refer to book for the definitions and

Unit Symbol In Liters formulas: page xv – xx in Worktext in
Kiloliter kL 1000 L Molecular Biology and Diagnostics
Hectoliter hL 100 L (2021) by Pineda, M.R., et.al.)
Dekaliter daL 10 L
Liter L 1L
Deciliter dL 0.1 L
Centiliter cL 0.01 L
milliliter mL 0.001 L

Volume Range Name/Title

0.2 – 2 uL P2
0.5 – 10 uL P10
2 – 20 uL P20
5 – 50 uL P50
10 – 100 uL P100
20 – 200 uL P200
100 – 1000 uL P1000
500 – 5000 uL P5000
1000 – 10000 uL P10000

LABORATORY MATHEMATICS
Terms:
✓ Molarity
✓ Molality
✓ Normality
✓ Percent Solution
✓ X solution
✓ Ratio
✓ Dilution
o Simple Dilution
o Serial Dilution
✓ Concentration
✓ Volume
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