Biotechnology Q1 Week 3 Students PDF

STE
Biotechnology
Quarter 1
Biological Techniques
STEM
STE
SSES
Learning Toolkit No. 3

The ASTRAL Project
Appropriate Science and Technology Resources for the Advanced Learners
Special Curricular Program in Science
DIVISION OF NEGROS OCCIDENTAL
Special Program in Science, Technology and Engineering

Department of Education
Special Science Learning Toolkit No. 3
BIOTECHNOLOGY
Special Program in Science, Technology and Engineering

Special Science Learning Toolkit 3
BIOTECHNOLOGY
Quarter 1: Biological Techniques
First Edition, August 2020
Republic Act 8293, section 176 states that: No copyright shall

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Published by the Department of Education—Division of Negros Occidental

Schools Division Superintendent: Marsette D. Sabbaluca, CESOVI
Chief Education Supervisor: Zaldy H. Reliquias
Education Program Supervisor: Dannie Clark M. Uguil
Development Team of the Module

Author: Filomena J. Bantigue
Management Team: Marsette D. Sabbaluca
Zaldy H. Reliquias
Dannie Clark M. Uguil
Department of Education – Division of Negros Occidental

Office Address: Cottage Road, Bacolod City, Philippines, 6100
Telefax: (034) 435-3960, (034) 703-3034
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This instructional material was collaboratively developed and

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or/universities. We encourage teachers and other education stakeholders to
email their feedback, comments, and recommendations to the Department
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We value your feedback and recommendations.
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Learning Competency:
Discuss the proper ways of handling the Autoclave, Balance, Pipette and
Burette.
What I Need to Know

When taking scientific measurements, it is important to be both accurate
and precise. Accuracy represents how close a measurement comes to its true
value. This is important because bad equipment, poor data processing or
human error can lead to inaccurate results that are not very close to the truth.
Who says science is boring, you just need to devote some time to realize
how fascinating it can be. “Nothing in life is to be feared, it is only to be
understood. Now is the time to understand more, so that we may fear less.” -
Marie Curie
This toolkit will guide you on how measuring equipment is often one of
the biggest outlays in a laboratory so taking adequate care of what you have
(therefore preventing unnecessary re-purchases) is an added bonus.
After going through this learning toolkit, you are expected to:
1. identify the purpose of an autoclave, pipette, balance, and burette;
and
2. discuss the roper ways of handling these laboratory apparatuses.
What is It
NAME IT, PAIR IT!

NAME the laboratory instruments and
PAIR it with its purpose/ use.
Used for measuring and transferring quantities of liquids from one container
to another.
Used for titration in volumetric analysis.
Used to sterilize surgical equipment, laboratory instruments, pharmaceutical
items, and other materials.
Designed to measure small mass in the sub-milligram range
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2
1
3 4
For a successful experiment, sanitation, disinfection, proper handling of

laboratory apparatus is very important.
Write LIKE if the PICTURE denotes proper handling of equipment/ apparatus.
Write DISLIKE if the PICTURE denotes improper way of handling equipment
or apparatus.
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1. 2.
3. 4.
5. 6.
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Who invented the following

lABORAtory AppArATus?
MATch the inventors from the
box below with their inventions.
Write the NAme of the APPARatus
AND the inventor on A piece of
pAP er.
Joseph Black Heinrich Schnitger
What is It
Charles Chamberland Francois Antoine Henri

Descroizilles
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Original image of apparatus made by inventors
1. 2.
Analytical balance (Lab Balance) Autoclave
3. 4.
Micropipettes Buretta or Burette
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What is it?
Sterilization
Sterilization is a term referring to any process of that removes or kills all
forms of microbial organisms such as fungi, bacteria, viruses, and spore forms,
etc., present in a surface, contained in a fluid, or in a compound such as
biological culture media.
Sterilization can be achieved by applying heat, chemicals, irradiation,

high pressure and filtering.
Autoclave is an equipment used to remove microorganisms (bacteria,

virus, fungus etc) and spores by high pressure, and high temperature steam
sterilization.
Auto meaning self; and clavis is self locking.
The invention of the autoclave

sterilizer is attributed to Charles
Chamberland, in 1879. Around
that time, researchers started to
understand the advantages of sterile
surgery, and doctors needed a more
reliable sterilization method than open
flaming.
The autoclave’s benefits were soon Charles Chamberland
evident, and it became an essential with his autoclave
part of every clinic and hospital.
An autoclave is also used to sterilize surgical equipment, laboratory

instruments, pharmaceutical items, and other materials. It can sterilize solids,
liquids, hollows, and instruments of various shapes and sizes. Autoclaves vary
in size, shape and functionality. A very basic autoclave is similar to a pressure
cooker; both use the power of steam to kill bacteria, spores and germs resistant
to boiling water and powerful detergents.
It is also a pressurized device used to heat aqueous solutions above
their boiling point at normal atmospheric pressure to achieve sterilization.
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Autoclave Safety Procedure
Autoclaves use high pressure and high temperature steam to kill

microorganisms and render biohazardous material inactive. For effective
sterilization, the materials/load must be saturated with steam. Air pockets or
insufficient steam supply will prevent effective sterilization. Proper cycle
parameters for effective decontamination of infectious waste are done using
autoclave indicators and performing autoclave validations.
Potential risks of using an autoclave are:

1. Heat and steam burns, hot fluid scalds, injuries to hands and arms
from the door, and bodily injury in the event of an explosion.
2. Exposure to biohazardous material may occur if biohazardous waste
is improperly packaged or manipulated. Onsite training on how to use
the autoclave properly and safely is essential for all new employees
to prevent injury. The use of heat-insulating gloves, lab coat, and
closed-toe shoes help prevent burns and scalds during loading and
unloading the autoclave.
The following can prevent injuries in the use of autoclave:

 Wearing appropriate Personal Protective Equipment (PPE) including
a lab coat, heat resistant gloves, and eye protection, especially when
 Never sealing containers; under pressure they pose an explosion
risk.
 Never opening the door to the autoclave if there is water running out
the bottom. Clogged steam lines, equipment malfunction, or
plugged drains may cause a buildup of scalding water.
 Waiting for the pressure to reach zero and the temperature is at or
below 121°C before opening the door at the end of a cycle to avoid
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steam burns and shattered glassware. Do not stand directly in front
of the door.
 Never superheating liquids. Superheating is a condition that occurs
when liquids are at a temperature above their normal boiling point but
do not appear to be boiling. Any disturbance of the liquid could cause
some of it to violently flash to steam and spraying. In situations where
personnel are in a hurry to remove flasks or bottles from the
autoclave, the superheated liquids may boil out of their containers or
explode.
Never autoclave the following:

 Sharps: It is not necessary to autoclave discarded sharps (used/unused
needles and syringes, contaminated broken glass, microscope slides
and coverslips, Pasteur pipettes, scalpel or razor blades) prior to
disposal in a sharps disposal container.
 For a pickup of full sharps containers, fill out the online form.
 Hazardous chemicals (including items contaminated hazardous
chemicals). Do not autoclave flammable, reactive, corrosive, or toxic
chemicals (e.g., alcohols, chloroform, acetic acid, formalin, or fixed
tissues). Lab coats that have been contaminated with chemicals should
not be autoclaved but cleaned by an approved laundry service or
disposed of as chemical waste.
 Dried bleach and bleach-associated materials, or nitrocellulose; both
compounds pose a fire or explosion risk.
 Radioactive materials: Contact the DRS Radiation Safety Program for
information on proper disposal of radioactive materials.
 Pathological waste: Includes animal carcasses, tissues, and organs and
human tissues and organs. University policy requires that certain types
of pathological waste be disposed of by incineration.
 Low Molecular Weight (LMW) biotoxins and prions: Some biohazards
will not be inactivated by autoclaving, as the material is extremely stable.
Preparation of Materials
To ensure adequate steam penetration, pack solid materials loosely; do
not intentionally compact waste or overfill biohazardous waste bags.
Bags/containers should be placed in a large, leak-proof, non-glass, shallow pan
to contain spills. Stainless steel pans or plastics that can be autoclaved
repeatedly at high temperatures (e.g., polypropylene, polypropylene copolymer
or fluoropolymers) are recommended.
Before processing, open the bags/containers so that the steam can

penetrate and effectively raise the temperature for adequate sterilization. A
small amount of water may be added to ensure heat transfer inside the
bag/container. If the bag is closed during autoclaving, the temperature of the
contents may not be raised sufficiently for decontamination. If processing more
than one tray, make sure that there is ample room between the trays so as to
not impede steam circulation.
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Place containers of liquid (e.g., bottles, beakers, flasks) topped with a cotton
plug or steam-penetrable bung in a large, leak-proof, shallow pan. Inspect the
glass to make sure there are no cracks.
Do not fill containers to the top, and leave plenty of head room. Bottles
with narrow necks may boil over if filled too full. Avoid the use of bottles if
possible, but if it is necessary, make sure that the screw-cap is nearly
unscrewed to allow for pressure changes or it may explode. Water should be
added to the pan to help prevent heat shock to the containers.
Basic Operating Instructions

The following are basic instructions for autoclave use but do not replace
the manufacturer's operating instructions and hands-on training. Before using
any autoclave for the first time, read and thoroughly understand the owner's
manual because many makes and models have unique characteristics.
1. Place the items to be autoclaved in the chamber.
2. Check the drain screen to make sure that it is not plugged or obstructed.
3. For bench top units that do not have inline steam, check and fill the
reservoir with de-ionized water (has been treated to remove all ions) to
the fill line (see manufacturer's instructions).
4. Close and seal the autoclave door.
5. On the autoclave keypad, or dials, select:
a. The type of load: gravity or liquid.
b. The sterilization time: The sterilization time will vary according to
the contents and how the load is packaged and should be
measured after the temperature of the materials reaches 121°C
and 15 pounds per square inch (PSI). It can be variable with a
minimum of 12-15 minutes. Several trays with large bags or
containers loaded in the autoclave will require a longer time to
reach 121°C and should be set accordingly.
c. The sterilization temperature. Unless specifically instructed, the
chamber temperature is set to 121°C (250°F).
d. A dry cycle if desired.
e. Or select a preprogrammed cycle, i.e,. the “waste” cycle if your
autoclave has this option. Preprogrammed cycles are either
entered at the factory or by the autoclave responsible party.
6. Run the autoclave cycle. Fill out the autoclave log if this is required.
7. At the completion of the cycle, don appropriate PPE before opening the
door. Wear safety glasses, a lab coat with long sleeves, closed-toed
shoes, and heat-resistant gloves.
8. Open the door slowly and only slightly and allow steam to escape.
9. Allow items to cool in the autoclave for at least 10 minutes before fully
opening the door.
10. Check the autoclave tape for a color change, and the print-out from the
recorder to see if the time and temperature were attained. If not, the load
should be re-autoclaved in another autoclave.
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11. Any bag displaying the biohazard symbol must be over bagged with an
opaque trash bag and sealed prior to disposal in the regular waste
stream. Bags with the biohazard symbol, regardless of use, must not be
placed in the regular waste stream without being over bagged.
Guide to Temperatures and Times

Biological
Glassware
Waste Liquids Dry Items
Items (Gravity
(Gravity (Liquid Cycle) (Gravity Cycle)
Cycle)
Cycle)
Open the
Loosen caps or
bag >2", Dirty: Place in
use a vented Fabrics Wrap;
Place in middle of the
closure, Instruments:
Preparation tray, pan;
Fill containers Clean, dry, lay in
Place Clean: wash,
no more than pan
indicator if rinse, wrap
75% capacity
needed
Dirty: In
Fabrics: Separa detergent and
Placement In the ted, on edge; pan;
Upright in pan
in Autoclave center Instruments: Clean: On
Flat side or
inverted
Temperature 121°C 121°C 121°C 121°C
60-120
min. 22 min. for
Treatment depending volumes
Time in on load <100mL; 30-60 min. 30-60 min.
Minutes size and 40 min. for
packing volumes >100mL
density
Dirty: Slow
Exhaust Slow Fast exhaust
Slow exhaust exhaust; Clea
Cycle exhaust and dry
n:Fast/dry
Avoid
puncturing Glassware
Check reference
bags. Hot bottles may with cracks or
for proper
Notes: Overbag explode. Let cool deep
packaging
and before moving. scratches may
methods
dispose of crack
properly.
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Proper Use of Balances

Laboratory balances from a general standpoint measure the mass of an
object, in the laboratory they are used to measure solids, liquids, tissue, they
have a wide range of uses in virtually any laboratory including
clinical, research and environmental settings.
Different types of balances are selected according to the function they

must perform. Laboratory balance types include toploading balances,
portable balances, analytical balances, semimicrobalances, and
microbalances. There are some overlaps in terms of readability and accuracy.
1. Top Loading Balance
A top loading balance (also referred to as

toploader balance) is among the most common types
of weight measuring scales used in the laboratory.
Top loading balances are available in a variety

of sizes and weight capacities, from 20 g to 64.1 kg.
Compact top loading balances are calibrated with fully
automatic time and temperature-controlled
adjustment, an internal or external weight, or clock
calibration.
A top loading balance can be tailored towards specific applications (i.e.

pipette calibration balances). Particular features include computer interface
capability via USB or RS-232C ports, short response times of 0.8 seconds or
less, and security measures to prevent illicit use of the scale.
2. Portable Balance
Portable balance can be used to
describe any digital weighing balance that can
be moved around. The term portable scales is
used to describe a weighing scale that can be
powered by batteries without depending on a
mains connection.
3. Analytical Balance
Analytical balances are highly sensitive lab
instruments designed to accurately measure mass.
Their readability has a range between 0.1mg -
0.01mg. Analytical balances have a draft shield or
weighing chamber to prevent the very small samples
from being affected by air currents.
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The modern two-pan analytical balance (lab balance)
was invented by Scottish chemist Joseph Black.
4. Semimicrobalance and microbalance
Semimicrobalance Microbalance
Can weigh to one hundredth of a milligram. They are generally used for
extremely specialized applications, such as differential weighing of a sample
before and after incineration, measurement of coatings, or weighing chemically
sensitive samples inside a glove box.
What is The Difference Between a Scale and a Balance?

Scales and balances are both weighing machines, however the
difference between a scale and a balance is that a weighing scale measures
weight relevant to the force of gravity, while a weighing balance is used to
compare the mass of two different objects.
What is a weighing scale?

Historically a weighing scale was a device that displayed weight by
measuring a deflection (turning,diversion), such as a spring scale. In modern
weighing machines, scales generally use springs or strain gauge load cells. It
is commonly accepted that a scale is less sophisticated in application and
precision ( being exact or accurate) than a balance and is typically used within
food, health, industrial and commercial industries for weighing ingredients,
monitoring personal health in gyms or at home, and for check weighing or
stocktaking procedures for businesses.
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RULES FOR ANALYTICAL BALANCES

The following rules summarize those procedures which must be followed
in order to obtain accurate and reliable mass measurements with a single-pan
analytical balance. Adherence to these rules will, at the same time, prevent
damage to the balance.
1. Close the balance door, while weighing an object, in order to prevent air
currents from disturbing the reading. When finished, the operator should
close the balance door to prevent dust and dirt from entering the
balance.
2. Only glass, ceramic, metal or plastic objects and containers should be
placed in direct contact with the balance pan.
3. Do not handle objects to be weighed with bare hands. Moisture, grease
and dirt on you fingers will affect the weight of the objects.
4. To be weighed accurately, all objects must be at room temperature. A
warm object sets up convection currents inside the balance enclosure,
which will make an object appear lighter than it really is. Also, warm air
inside the enclosure is less dense than the air that it displaces and this
also leads to a negative determinate error.
5. Never weigh chemicals directly in contact with the balance pan. Use
containers such as beakers, flasks and weighing bottles.
6. All objects and materials that have recently been removed from a
desiccator will absorb moisture and thereby gain weight. It is therefore
good practice to record weights after identical time intervals. For
example if you are taking crucibles to constant weight. Always record the
weight of the crucible exactly 5 seconds after having placed the crucible
on the balance pan. Using this technique it is possible to minimize the
effect of moisture absorption.
7. The use of weighing paper must be strictly avoided when using an
analytical balance.
8. Do not spill chemicals inside the balance enclosure. If a spill occurs,
clean it up immediately.
Steps in using Analytical Balance in a Laboratory

1. First, before weighing anything on this
analytical balance, it needs to be "tared,"
or recalibrated to read 0.0000 g. When
first turned on, or when left by the
previous user, the balance may indicate
something other than 0.0000 g. The Tare
button needs to be pressed and
released to effect this recalibration.
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The four images below illustrate this process.
2. An analytical balance is so sensitive that it can detect the mass of a

single grain of a chemical substance. Thus, if a method of direct
weighing is used, the substance ought to be added to the tared container
which will hold it, NEVER directly to the pan or even to weighing paper
placed on the pan.
3. The container used should be completely dry and at room temperature,
never at an elevated or reduced temperature. Even slight temperature
differences can produce APPARENT changes in mass of the container.
4. Finally, the container ought to be completely dry, inside and out. All that
having been said, here are some images showing various correct ways
of carrying out weighing using an analytical balance.
Regardless of which method illustrated below is used accurately to

weigh a sample, the sample, placed in a weighing bottle set in the upturned cap
in a beaker with a watch glass placed on top, must be first dried in the oven.
You may identify your sample by marking the beaker but DO NOT mark the
balance.
The ovens are kept at 110 0C, but our

ovens may show slightly lower
temperatures owing to the doors being
opened repeatedly during normal laboratory
activities.
The tried-and-true method of transferring a

precisely weighed sample uses "weighing
by difference," shown here. The empty
balance is tared, then the weighing bottle
with cap is placed on the pan and weighed
to ±0.0001 g.
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The weighing bottle is

removed in a manner which
avoids the transference of oil
or other matter from one's
fingers.
The cap is likewise removed

from the bottle.
The weighing bottle is tipped above the container to receive the sample and a
small amount is allowed to fall out of the weighing bottle.
The weighing bottle is tipped back up and tapped gently to make sure all of
the substance falls back in the bottle and doesn't remain on the bottle rim. The
cap is replaced and the bottle weighed once again. The difference between
the first and second weighings represents the amount transferred.
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Using the Top Loading Balance

Several top loading balances, precise to ±0.001 g, are available for your
use in the balance room. Many professors of Quantitative Analysis do not allow
them to be placed in the same room where the analytical balances are kept
because students often use them to save time when they ought to be using the
analytical balances.
First of all, the top loading balances are less precise by a factor of 10
and secondly, air currents around the pan can reduce that precision by as much
as another factor of 3 or 4. But the top loading balance is the instrument of
choice where precision is not of great importance. Here is one our top loading
balances. It can be "tared" by pressing the front bar, as shown below.
In the drawer below the top loading balances

you will find weighing paper. This paper must be
used ONLY on the top loading balances,
NEVER on the analytical balances, because
there is always the chance that some of the
substance being weighed will stick to the
weighing paper after the weight has been
recorded thus producing an error on the low
side.
Once a piece of weighing paper has been

placed on the pan, and the balance set to 0.000
by taring it, a sample can be removed from the
container holding it and placed on the pan,
repeating the process as many times as are
necessary until the weight of material needed is
achieved, as shown here.
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How to use the pipette?

Pipettes are small tools used in laboratories for measuring and
transferring small amounts of liquid. They come in several sizes and shapes,
with various features and functions. Pipettes help scientists, doctors, and
researchers perform experiments and tests by accurately measuring liquid
volumes. They must be handled with care and cleaned properly.
Part 1: Drawing up of Volumes of liquid with pipette
Step 1
Use pipettes to accurately
measure small volumes of
liquid.
Step 2
Keep the pipette tip away from the
bottom of the vessel.
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Step 3
Draw up liquids
with reverse
pipetting
technique.
Step 4
Release liquid by
reverse pipetting
technique.
Part 2: Use pipettes to disperse volumes of liquid.
Step 1:
Disperse volume of liquid with
pipettes.
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Step 2
Measure volumes
accurately.
Step 3
Use the forward pipetting technique.
Step 4
Disperse the liquid by forward pipetting
technique.
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Part 3: Cleaning Pipettes
Step 1
Clean the outside with mild soap.
Step 2
Clean the interior with distilled
water and alcohol.
Step 3
Use a strong detergent to rinse
off radioactive substances.
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Step 4
Boil pipette pieces and rinse
them with water to remove
nucleic acids.
Step 5
Use a strong detergent solution
to rinse off protein particles.
Source : Creative Commons
Step 6
Types of Pipettes
Allow to fully air dry before
reusing.
Types of Pipette
1. Volumetric Pipette, bulb pipette or belly pipette - allows extremely
accurate measurement (to four significant figures) of the volume of a
solution. It is calibrated to deliver a fixed volume of a liquid.
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 Transfer pipette- are disposable plastic pipets used to transfer small

volumes of liquids. Transfer pipets are also called Pasteur pipets,
teat pipets, droppers, eye droppers and chemical droppers.
Eye Dropper or Pasteur Pipette

1. Measurement or Graduated pipettes
 Mohr graduated Pipette - provide an accurate way of delivering small
volumes of liquids.The glass has permanent graduations in a descending
scale for ease of use. Color coded for easy identification.
Pyrex Color -coded Mohr Pipette, 2mL,with .01 graduation
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 Serological Pipette is a nearly ubiquitous (found everywhere) laboratory

instrument used for transferring milliliter volumes of liquid. Serological
pipettes typically have graduations along their sides for measuring the
amount of liquid being aspirated or dispensed.
What is the difference between a Mohr pipette and a serogical pipettes?

A Serological pipette is a graduated pipette in which the calibration marks
extend all the way to the tip. A is a graduated pipette in which
the calibration marks do not extend to the tip but at a point above the tip.
Source:SlidePlayer
 Micropipettes -utilized in the laboratory to transfer small quantities of liquid,
usually down to 0.1 uL. They are most commonly used in chemistry,
biology, forensic, pharmaceutical, and drug discovery labs, among others.
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Oxford micropipettes Gilson micropipettes

Pipettes and micropipettes are used to measure and deliver accurate
volumes of liquid. The difference between the two is
that micropipettes measure a much smaller volume, starting at 1 microliter,
while pipettes generally start at 1 milliliter.
Burette/Buret
What are burettes for?
Burette, also spelled Buret, laboratory apparatus used in

quantitative chemical analysis to measure the volume of a liquid or a gas.
A buret is a piece of volumetric glassware used to deliver variable liquid

amounts typically for titrations. It is a long glass cylinder open at one end and
fitted at the opposite end with a stopcock valve (Figure 1). The stopcock valve
controls the flow of liquid from the cylinder through the buret tip.
Figure 1.
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Francois Antoine Henri Descroizilles invented the first burette in 1791.
There are two main types of burette

1. Volumetric burette
A volumetric burette delivers measured volumes of liquid.

-which is used in analytical chemistry for accurate dispensing
of variable, and for measuring the volume of a liquid,
especially of one of the reagents in a titration.
Glass Acid Burette,

Volumetric Burette
2. Piston burettes are similar to syringes, but with a

precision bore and a plunger.
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Proper use of Buret

Storing Buret Tubes
First of all, you MUST learn how to put your buret away properly in the
buret cabinet. Here's a picture of the buret cabinet on the left. On the right we
have three burets stored properly. Note the arrows pointing to the holes into
which one "ear" of the petcock valve is placed. Placing the buret in the cabinet
this way assures that the buret will not fall out when the cabinet is opened.
The "petcock" or draining valve which allows titrant to flow out the nozzle
is made of teflon. On one end is a handle for opening and closing it. On the
other is a tightening nut, a rubber o-ring and a teflon spacer. Your buret ought
to be assembled as shown in the photo at the left with the teflon spacer rubbing
against the buret and the rubber o-ring between the spacer and the nut.
Otherwise there is a tendency for the nut to loosen with repeated turns of the
petcock.
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How to Read Buret Tubes

When reading a buret it is important that your line of
sight be in a direction perpendicular to the buret column.
Note in this photograph that although the bottom of the

meniscus is clearly outlined, the variability of the
background does not always offer such visibility.
More likely than not, the bottom of the mensicus is

lightened by random reflections in the laboratory. Such
variability can produce errors of several hundredths of a
milliliter. All buret reading should be done using a buret
card.
Source:
http://www.phschool.com/science/biology_place/labbench/lab2/analysis.html
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The black streak is produced

using a felt tipped pen and offers
the student a constant dark
reflection against a white
background for higher precision in
determining relative titrant
volumes.
Another problem often encountered by students is

poor drainage.
Note that this buret has droplets which stick to the

inner wall. If your buret shows such droplets, use one
of the buret brushes and Alconox to wash the inner
surface.
If that doesn't improve the drainage, ask your

instructor to draw some dichromate/sulfuric acid
cleaning solution into the buret for more thorough
cleaning.
A 50 mL buret can be read to ±0.01 mL, but in order to be able to

interpolate (estimation) to the last digit, the perpendicular line of sight must be
followed with meticulous care.
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Note in these two photographs, one in which the line of sight is slightly
upward and the other in which it is downward, that an interpolation is difficult
because the calibration lines don't appear to be parallel.
The use of a buret card and a line of sight perpendicular to the buret column
are techniques which must be adopted to achieve maximum precision. Note the
final photograph in which the level of the meniscus bottom can be determined
to within ±0.01 mL. What reading would you report for this buret volume?
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A buret card ought to have a black streak with a distinct horizontal zone
of black against white. Moreover, when held behind the buret, the upper limit
of the black streak ought to be placed just under the meniscus, so that the
bottom of the meniscus can be seen distinctly against a narrow zone of white.
Note in the photo at the left that the apparent level of the meniscus is
different from that on the right. Since placing the black streak just under the
meniscus is more repeatable than at some variable distance, the close
placement is recommended.
If you are assigned a buret with a white instead of a blue or red scale,
a buret card other than white ought to be used. Notice in the photo at the left
the white scale is rendered invisible by the white card but can be seen more
clearly with the pastel blue card on the right.
32
BIOTECHNOLOGY
Cleaning Buret Tubes
Pipettes and burettes accumulate inert solid material which must be
removed from time to time. Here at the left is the nozzle of a burette which has
material which will not pass through.
You may have to use a wire, available on the lower ledge of the burette
case, to clean out this material. It is best to do it with the petcock valve removed
so that when you do a reverse wash after poking it free, the material can be
washed out at the point of the valve instead of at the other end of the burette
cylinder.
A bubble in the nozzle of a buret will produce an inaccurate volume

reading if the bubble escapes during a titration. Bubbles may be large
and visible as shown above left or so small as not to be seen, above
center.
During a titration such small bubbles begin to move in the direction

of the nozzle but may remain in place even though there is a moderate
flow of titrant (above right). Even when the buret valve is wide open
some bubbles remain in place until you take your eyes off them. Then
they sneak through the nozzle and ruin your titration.
33
BIOTECHNOLOGY
The quickest way to get rid of nozzle bubbles is to fill the buret with titrant and
open the valve. The pressure of the titrant in a full buret is often enough to force
all bubbles out.
If that doesn't work, two other methods can be used. The first is to use some
chaotic suction. The student on the left has first rinsed then filled her buret with
titrant. Her attempt to force out all bubbles by the first method didn't work, so
she opens the buret valve, letting the contents begin to drain into a beaker. She
uses her bulb momentarily to suck air through the nozzle.
The mixture of small bubbles which are produced reenter the nozzle. The chaos
and production of small bubbles (right) is often capable of driving out all bubbles
from the nozzle.
If that doesn't work, immerse the nozzle of the buret in a small beaker of titrant
and while using the bulb to reverse the flow of titrant, open the valve (right).
The bubble will emerge from the top of the valve and rise to the top of the
column of titrant, but no air will be able to enter the mouth of the nozzle.
Finally, a brand new buret, just taken out of the box, ought to be examined
carefully, as ought a buret returned to service after having been in storage a
long time. Production errors are rare but they do occur. Burets in storage may
be there for reasons not connected with a lack of need.
34
BIOTECHNOLOGY
What’s More ?
Worksheet 1: Proper Handling of an Autoclave

Fill in the table with the correct answer. May insert rows for additional answers.
Potential Things not Basic
Autoclave Autoclave
risks of using allowed to operating
safety Preparation
an autoclave autoclave instructions
1. 1. 1. 1. 1.
2 2. 2. 2. 2.
3. 3. 3. 3.
4. 4. 4. 4.
5. 5. 5. 5.
6. 10. 10.
Worksheet 2: Proper Handling of Balance

1. List down the rules to be followed in order to obtain accurate and reliable
mass measurements with a single-pan analytical balance. Adherence to
these rules will, at the same time, prevent damage to the balance.
2. State the ways of using Top Balance.
3. Write your answers on a piece of paper.
Write your answers on a piece of paper.

1.
What are the risks of using an autoclave?
35
BIOTECHNOLOGY
2.
Autoclave Safety
How can we
prevent injuries
while using an
autoclave?
3.
Never autoclave
the following
materials.
What are the steps in preparing materials for

autoclave?
4.
36
BIOTECHNOLOGY
Direction: Write GO if the statement /illustration is CORRECT and STOP if

the statement/illustration is INCORRECT.
1. The invention of the autoclave sterilizer is attributed to Charles
Chamberland, in 1879. Around that time, researchers started to
understand the advantages of sterile surgery, and doctors needed a
more reliable sterilization method than open flaming.
2. Exposure to biohazardous material may occur if biohazardous waste is
improperly packaged or manipulated. The use of pot holder, lab coat,
and closed-toe shoes help prevent burns and scalds during loading and
3. Always superheating liquids. Superheating is a condition that occurs
when liquids are at a temperature above their normal boiling point but do
not appear to be boiling. Any disturbance of the liquid could cause some
of it to violently flash to steam and spraying.
4. To ensure adequate steam penetration , pack solid materials loosely; do
not intentionally compact waste or overfill biohazardous waste bags.
Bags/containers should be placed in a large, leak-proof, non-glass,
shallow pan to contain spills. Stainless steel pans or plastics that can be
autoclaved repeatedly at high temperatures (e.g., polypropylene,
polypropylene copolymer or fluoropolymers) are recommended.
5. Biological waste should be placed in the autoclave onside or inverted
with the temperature of 1210C
6. Scales and balances are both weighing machines, however the
difference between a scale and a balance is that a weighing balance
measures weight relevant to the force of gravity, while a weighing scale
is used to compare the mass of two different objects.
7. Close the balance door, while weighing an object, in order to prevent air
currents from disturbing the reading. When finished, the operator should
close the balance door to prevent dust and dirt from entering the
balance.
8. Never weigh chemicals directly in contact with the balance pan. Use
containers such as beakers, flasks and weighing bottles.
9. Before weighing anything on this analytical balance, it needs to be
"tared," or recalibrated to read 0.0001 g.
10. Weighing paper must be used ONLY on the analytical balances, NEVER
on the top loading balances, because there is always the chance that
some of the substance being weighed will stick to the weighing paper
after the weight has been recorded thus producing an error on the low
side.
37
BIOTECHNOLOGY
Draw up liquids with reverse

pipetting technique.
11.
12
Measure volumes accurately.
13.
Clean the interior with distilled

water and alcohol.
14. Volumetric Pipette , bulb pipette or belly pipette - allows extremely accurate
measurement ( to four significant figures) of the volume of a solution. It is
calibrated to deliver a fixed volume of a liquid.
15. Pipettes and micropipettes are used to measure and deliver accurate
volumes of liquid. The difference between the two is
that micropipettes measure a much smaller volume, starting at 1 microliter,
while pipettes generally start at 1 milliliter.
38
BIOTECHNOLOGY
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Learning Toolkit No. 3

Special Curricular Program in Science

DIVISION OF NEGROS OCCIDENTAL

Special Program in Science, Technology and Engineering

Special Program in Science, Technology and Engineering

Republic Act 8293, section 176 states that: No copyright shall

Borrowed materials (i.e., songs, stories, poems, pictures, photos,

Published by the Department of Education—Division of Negros Occidental

Development Team of the Module

Department of Education – Division of Negros Occidental

This instructional material was collaboratively developed and

We value your feedback and recommendations.

What I Need to Know

NAME IT, PAIR IT!

For a successful experiment, sanitation, disinfection, proper handling of

Who invented the following

Joseph Black Heinrich Schnitger

Charles Chamberland Francois Antoine Henri

Original image of apparatus made by inventors

Analytical balance (Lab Balance) Autoclave

Micropipettes Buretta or Burette

Sterilization can be achieved by applying heat, chemicals, irradiation,

Autoclave is an equipment used to remove microorganisms (bacteria,

The invention of the autoclave

An autoclave is also used to sterilize surgical equipment, laboratory

Autoclave Safety Procedure

Autoclaves use high pressure and high temperature steam to kill

Potential risks of using an autoclave are:

The following can prevent injuries in the use of autoclave:

Never autoclave the following:

Before processing, open the bags/containers so that the steam can

Basic Operating Instructions

Guide to Temperatures and Times

Proper Use of Balances

Different types of balances are selected according to the function they

1. Top Loading Balance

A top loading balance (also referred to as

Top loading balances are available in a variety

A top loading balance can be tailored towards specific applications (i.e.

4. Semimicrobalance and microbalance

What is The Difference Between a Scale and a Balance?

What is a weighing scale?

RULES FOR ANALYTICAL BALANCES

Steps in using Analytical Balance in a Laboratory

2. An analytical balance is so sensitive that it can detect the mass of a

Regardless of which method illustrated below is used accurately to

The ovens are kept at 110 0C, but our

The tried-and-true method of transferring a

The weighing bottle is

The cap is likewise removed

Using the Top Loading Balance

In the drawer below the top loading balances

Once a piece of weighing paper has been

How to use the pipette?

Part 1: Drawing up of Volumes of liquid with pipette

Part 2: Use pipettes to disperse volumes of liquid.

Part 3: Cleaning Pipettes

Source : Creative Commons

 Transfer pipette- are disposable plastic pipets used to transfer small

Eye Dropper or Pasteur Pipette

 Serological Pipette is a nearly ubiquitous (found everywhere) laboratory

What is the difference between a Mohr pipette and a serogical pipettes?