Practical No.

– 01

SCT 221-1
MICROBIOLOGY
Microbiology Laboratory Equipment and Instruments and
Aseptic Techniques

UWU/SCT/20/035
Rameesh Ishak
2022.09.27
Date – 2022.09.22

Practical No. – 01

Practical Title – Microbiology Laboratory Equipment and Instruments and

Aseptic Techniques.

Objective – To provide hands on experience on various microbiological laboratory equipment

and instruments.

Introduction – In this practical, we observed and studied various important laboratory tools
and equipment utilized in microbiology. They were,

 pH meter
 Magnetic stirrer
 Analytical balance
 Pipette and Pipette filler
 Micropipette
 Laminar flow cabinet
 Autoclave
 Bio safety cabinet
 Incubator
 Colony counter
 Hot air oven
 MC bottles
 Spreader
 Petri dishes
 Inoculating loop and needle
 a) pH meter

Lever

Display
Probe with the electrode

Keypad Solution to be tested for the pH

Figure 1

A pH meter is a precise device that measures the hydrogen-ion movement in suspensions made
of water and displays the acidity or alkalinity of said suspensions as pH.

 Lever – Used to dip the probe to the solution contained vessel.

 Display – Display the pH value of the solution.
 Keypad – To On/Off the display and to start the measure of pH.
 Probe with the electrode - An electrochemical potential is created when hydrogen ions
in the solution interact with positive charges on the electrode and is represented on a
display in terms of pH units.

Procedure: –

The pH meter was turned on and the electrode was cleaned by distilled water. Then it was
wiped by a tissue since no water must be present in the probe. Then to calibrate the pH meter
three buffer solutions with pH 7.0,4.0 and 10.0 were used. In each instance where the buffer
solutions were replaced to measure the pH, the electrode was rinsed using distilled water. After
the calibration was over the solution to be measured for pH was placed and the pH was
measured.
 b) Magnetic stirrer

Metal utensil

Magnetic Heating plate

Magnetic string bar

Heat adjuster

Stirring time adjuster Stirring speed adjuster

Figure 2

This device is usually used for mixing various liquid components in a mixture in a chemical
or microbiology laboratory. This is used in place of other stirrers are because the contamination
when stirring is less.

 Magnetic heating plate – to place the plastic container with the solution to be mixed or
mixed while heating.
 Magnetic string bar – a magnet which is used to place inside the solution which is
rotated when the equipment is ON due to magnetism.
 Stirring time adjuster – a timer to set the time required for the stirring.
 Stirring speed adjuster – a controller to control the speed of stirring.
 Heat adjuster – control the temperature of the magnetic heating plate.
 Metal utensil – used to take the magnetic string bar out from the solution by placing it
on the outer surface of the container.
Procedure: -
The power cord was connected, and the magnetic stirrer was switched ON. Then the container
with the solution needed for stirring was placed on the magnetic heating plate and the
magnetic string bar was immersed in the container. By the three knobs the stirring time, stirring
speed and the temperature was adjusted according to the requirements. After the stirring time,
first the heat was turned off and then the stirring speed was turned off. Then the metal utensil
was placed on the outer surface of the container and the string bar was taken out easily since it
sticked to the metal.
 c) Analytical Balance

Glass doors Balance pan

Display
Level adjustment feet Tear Button

Figure 3

An analytical balance measures the precision in determining the mass of solid objects, liquids,
powders, and granular substances. It is an electronic device and uses the principle of magnetic
force restoration, offering readability up to 0.0001 g. Since it is an enclosed equipment,
external factors like air effecting the reading of the weight to be calculate is very low.

 Glass Doors – open and close the container.

 Balance pan – the weighing board with the substances is placed on this.
 Level adjustment feet – to level the plane where the equipment is placed.
 Display – to display the weight of the substance.
 Tear button – to make the display weight zero.

Procedure: -
The balance level is checked (check that around the screen there is a bubble indicating the level
of the balance) If it is imbalance by the level adjustment feet the level was balanced. By
pressing the power button balance was turned ON. There should be nothing on top of the pan,
so at the beginning the measurement should be zero, if not by the tear button the display
weight was turned to zero. Then the weighing board with the substance to be weighted was
placed on the balance pan. The glass doors were tightly closed. The reading was taken, after
that the sample was taken out and the inside of the analytical balance was cleaned.
 d) Pipette and Pipette filler Pipette filler

Figure 4 E valve
Pipette S valve A valve
A pipette used to transfer a required amount of liquid from one vessel to another and to suck
the liquid to the pipette the pipette filler is used. In the pipette filler,

 A valve – to release the air inside.

 S valve – to siphon the liquid to the pipette.
 E valve – to eject the siphon liquid inside the pipette.

Procedure: -
First the pipette filler was fixed to the pipette, then the pipette was placed vertically on the
vessel with the liquid until the tip of the pipette was fully submerged, if the amount of liquid
present in the container is very little to fully submerge the tip of the pipette the vessel was
angled a little and the pipette was placed. The A valve was pressed to release the air inside then
the S valve was pressed to siphon the liquid to the pipette. Finally, the pipette was placed
above the required vessel and E valve was pressed to eject the liquid in the pipette.
 e) Micropipette

Push button

Tip ejector button Disposable Tips

Figure 5 Volumeter
Micropipettes are semi-automatic devices that draw and dispense liquid samples using
disposable pipette tips. In addition to getting an accurate measurement of the reagent and
sample, this helps reduce the possibility of human contamination in the lab.

 Push button – siphon the amount of liquid mentioned in the volumeter.

 Tip ejector button – eject the tip by pressing this button.
 Volumeter – adjust the required amount of volume.

Procedure: -
First the required volume was set in the volumeter and the tip was placed. The tip was placed in
the liquid and the liquid was siphoned by the push button when it was pressed once. Then to
another vessel, the sucked liquid was ejected again by same push button. To remove the
disposable tip the tip ejector button was pressed.
 f) Laminar Flow Cabinet

HEPA Filter

Fluorescent lamp UV lamp

Workstation

Figure 6 Control panel

Laminar Flow Cabinet is a closed device primarily for processes or instruments sensitive to
microbial contamination. It is used for experiments related to plant tissue culture and for the
experiments of genetic transformation. It is made up of stainless steel, avoiding joints and
corners to prevent the accumulation of bacterial spores.

 Workstation - a flat working station is present inside the cabinet for all the processes to
be taken place. Culture plates, burner and loops are all placed here.
 Control panel – control the ON/OFF functions of the fan, fluorescent light, UV light etc.
 Fluorescent lamp - provide proper light during the operation.
 HEPA filter - The High-efficiency particulate air filter is present within the cabinet that
makes the environment more sterile for the operation thus reducing the chances of
contamination.
 UV lamp – Produce UV rays that sterilizes the interior of the cabinet and contents before
the operation.
Procedure: -
Before running the laminar flow cabinet, the cabinet was checked to ensure that nothing
susceptible to UV rays is present inside the cabinet. Then the laminar flow cabinet was closed,
and the UV light was switched on. The UV light should be kept on for about 15 minutes to
ensure the surface sterilization of the working bench. The UV light was then switched off, and a
time of around 10 minutes was spared before the airflow was switched on. After the air flow
was switched on and 5 minutes was passed the cabinet was opened and the fluorescent light
was switched on to start the operation. To ensure more protection, the workstation was
sterilized with 70% ethanol. After the operation was finished the air flow, fluorescent light and
the glass door was closed/switched off.

g) Autoclave

Controls

Lid

Figure 7.1 Figure 7.2

Cooling system

Figure 7.3 Figure 7.4

Chamber (inside of the autoclave) Drip cage

The autoclave is an example of moist heat sterilization. The primary purpose of the autoclave is
sterilizing culture media and laboratory supplies. Saturated steam under pressure above 100℃
(about 120oC) is used for sterilization in autoclaves.

 Controls – switch ON the temperature and the instrument.

 Lid - seal off the outside atmosphere and create a sterilized condition on the inside of
the autoclave.
 Cooling system - Before the wastewater coming from the autoclave can enter the drain
piping, it must be cooled down to avoid damage caused by the heat.
 Chamber – main body of the autoclave where water is converted to steam.
 Drip cage – cultivation mediums, laboratory equipment are placed on this before
putting them inside the chamber.

Procedure: -

First a sufficient amount of water was put inside the chamber. Now, the materials to be
sterilized was placed inside the drip cage and the drip cage was placed inside the chamber. The
lid was then closed. To generate steam, temperature was risen. After the sterilization process
was finished by the pressure valve the steam was let out and after some time the lid was
opened to take out the sterilized equipment.

h) Bio safety cabinet

HEPA filter Control panel

UV light fluorescent light

glass door

Gas and water taps

Figure 8

Up foot switch Down foot switch

A microbiology laboratory deals with many infectious and hazardous organisms and different
carcinogenic chemicals. There is a very high risk of contaminating the environment and
personnel in the microbiology laboratory. Thus, the need for safe and contamination-free
transfer of specimens. The biological safety cabinet is the principal device used for containment
while dealing with microbiological samples.

 HEPA filter - The High-efficiency particulate air filter is present within the cabinet that
makes the environment more sterile for the operation thus reducing the chances of
contamination.
 Control panel – control the ON/OFF functions of the fan, fluorescent light, UV light etc.
 Fluorescent light - provide proper light during the operation.
 UV light – Produce UV rays that sterilizes the interior of the cabinet and contents before
the operation.
 Gas and water taps – taps which allow gas and water inside the cabinet.
 Up foot switch – red in color and by pressing it the glass door can move upward.
 Down foot switch – black in color and by pressing it the glass door can move downward.
Procedure: -

First the bio safety cabinet was turned ON. The UV light was turned ON to sterilize the
workstation for 15 minutes, after the UV light was turned off another 10 -15 minutes was
waited before opening the glass door for operation due being cautious of UV radiation. Then
the workstation was wiped off by 70% ethanol then the required experimentation or the
operation was conducted. Finally, after the experimentation the workstation was cleaned with
70% ethanol and the glass door was closed.

Control Panel buttons: -

Fan Glass door slide up

UV light Mute safety warning

Fluorescent light Power ON/OFF

Figure 9 Glass door slide down

 i) Incubator

Display

Temperature

Figure 10.1 Figure 10.2

It is an apparatus with a chamber used to provide controlled environmental conditions.

Temperature and humidity are adjusted to provide optimum conditions for the growth of the
microorganisms being handled. The temperature is critically regulated by the thermostat and
heating elements. Fan is fitted for circulation of air inside.

j) Colony counter

Hand lens Display

Controls
Glass grid

Figure 11
A colony counter is primarily used for counting the number of colonies present on a culture
plate to estimate the concentration of microorganisms in liquid culture. A Petri plate is placed
on an electronic pressure pad with light illumination, and each colony on the plate is marked by
tapping the plate with an auto marker probe pen. A count is registered in the digital display by
the touch’s pressure.
 Controls – turn on the lights and can reset the readings and can get the average.
 Glass grid – place the petri dish with micro-organisms colonies.
 Display – display the colony count.
 Hand lens – to see the colonies clearly.
Procedure: -
The instrument was turned on by pressing the on/off Switch. The Petri Plate was placed on the
Glass grid. The pen was pressed firmly keeping the Pen straight / vertical - on the Petri Dish
where a Bacteriological Colony was located. A count was registered by the counter, there was a
beep, and an ink dot were marked on the Petri Dish. This was continued till all the colonies
were counted. If there were many colonies, the petri dish was divided to equal sections and
colonies were counted in two opposite sections and the average was taken. This average was
multiplied by the number of sections in the petri dish to get an average reading on the no. of
colonies.

k) Hot air oven

Figure 12

Dry heat is used in a hot air oven to sterilize. Sterilizing glass objects like pipettes, flasks, steel
tools, and scissors is its primary use. Heat is produced in a hot air oven by electric current.
Because of how the heating components are arranged, the heat is distributed evenly. The
temperature affects the holding period. The equipment is typically used to dry metal
equipment, powders, waxes, and autoclaved materials. The primary parts of a dry heat sterilizer
are thermal insulation, continuous heat elements, a thermostat, a display, and perforated
metal sheets. For one to one and a half hours, this device is run at a temperature of 160 to 180
°C. The sterilizer is where wrapped goods are maintained.
 l) MC(McCartney) bottles

Figure 13

The McCartney Bottles are specially used to carry Culture Medium and bacterial growth can be
done in these bottles. McCartney Bottles are a set of thick, clear & wide mouth glass bottle with
an aluminium screw cap. These bottles along with the cap and rubber liner can be autoclaved
easily. Also, these bottles can be sterilized with the medium present inside it.

m) Spreader

Figure 14

To delicately spread cells and bacteria over a culture plate, such as a Petri dish, a cell spreader
or plate spreader is a manual tool used in biology and related sciences. There are many
different shapes and materials for cell spreaders, including glass, plastic, and metal. Each of
these materials has unique benefits and drawbacks. For frequent use, glass, for instance, is
easily sterilizable and on the other hand it is quite simple to shatter.
 n) Petri dishes

Figure 15

A petri dish is constructed of plastic or glass. It is a device into which culture media is poured,
creating an environment favorable for the development of microorganisms. Petri dishes are
made up of two identical components, but one of them is larger than the other and forms a
cap-like shape.

o) Inoculating loop and needle

Figure 16

Without introducing any undesired organisms, an inoculating loop and needle transfer
microbial growth from one container to another. Both types of equipment have a looped or
straight wire made of nichrome or stainless steel linked to a wooden handle.
Aseptic Techniques
Aseptic technique is a method that involves target-specific practices and procedures under
suitably controlled conditions to reduce the contamination from microbes. It is a compulsory
laboratory skill to conduct research related in the field of microbiology.
Material: -

 Five petri dishes

 Bunsen burner
 70% ethanol
 Tissues
 Incubator
 Para film
Procedure: -
The space where we conducted our studies was first carefully sanitized with 70% ethanol and
wiped with tissues. Before sanitizing the space, the Bunsen burner were preserved, and it was
placed as near together as feasible. They were positioned so that we had enough of room to do
our experiment. Then the five petri dishes were taken and from them three petri dishes were
taken one by one near the Bunsen flame and your thumbprint were placed on the agar medium
inside the petri dishes. Then they were covered by a parafilm for isolation, and they were
labeled as the date, group no, microorganism name if known, and if the thumbprint was
sterilized or not. This information was marked on the end or edge of the petri dish since you
need to observe the changes through the glass. Then two the remaining two petri dishes two
thumbprints that were sterilized by the 70% ethanol were placed and in the same manner the
two petri dishes were labelled. The five petri dishes were then kept in the Incubator at a
temperature of 300C to observe the changes.
Observation: -
A B C

Figure 17
A – Petri dish with a sterilized thumbprint with a little bit contamination.
B – Petri dish with an unsterilized thumbprint.

C – Petri dish with a sterilized thumbprint with unexpected contamination.

These are the observations that we observed.

Discussion: -
We found that one of the petri dishes with the sanitized finger was still contaminated and that
the results were not obvious, even though we followed the protocol to ensure that there are no
contaminants. If we had used the laminar flow cabinet, this may have been prevented, but we
were unable to do so due to a lack of resources.
We were able to notice that microbial colonies grew quickly because nutritional agar is nutrient
rich (within 4 to 5 days). This is because keeping the petri dishes in the incubator gave the
optimal temperature and conditions in addition to providing all the nutrients the
microorganism needed through the agar medium. Both factors contributed to the microbial
growth's rapidity.

Conclusion: -
The knowledge regarding the equipment and instruments in the laboratory and how the
mechanisms of it were updated. Also, the procedures to how to operate each of this equipment
and instruments were learned.
After conducting the experiment on aseptic techniques, we concluded that the aseptic
approach, when used in conjunction with appropriate sterilization procedures, can help us
avoid contamination when creating culture media. The laminar flow cabinet will assist us in
further avoiding contamination so that we can improve on what we did as agreed in the
conversation. As a result, we may draw the conclusion that the aseptic approach in
microbiology is a useful tool for growing microbial colonies with a relatively low level of
contamination.
References: -

 Shrestha, Ashma. “Equipment Essential for Microbiology Laboratory.” Microbe Online,

microbeonline.com/equipment-essential-for-microbiology-laboratory.

 “Instruments Used in Microbiology Laboratory - Principle and Uses | Biology Ideas.”

Biologyideas.com, 21 July 2021, biologyideas.com/instruments-used-in-microbiology-

laboratory/.

 “Cell Counters / Colony Counters | Labcompare.com.” Www.labcompare.com,

www.labcompare.com/Pharmaceutical-Lab-Equipment/421-Cell-

Counters/#:~:text=Colony%20counters%20count%20the%20number.

 AROUND LAB NEWS / EN» Application Note – SOP N.3 – Colony Counting: Standard

Operating Procedure for Colony Counter. www.aroundlabnews.com/en/application-note-

sop-n-3-colony-counting-standard-operating-procedure-for-colony-counter/.

 “Biosafety Cabinet (BSC): Introduction, Types, Handling Procedure.” Universe84a, 25

Oct. 2021, universe84a.com/biosafety-cabinet/.

 “Autoclave & Sterilizers.” Www.narulaexports.com,

www.narulaexports.com/en/products/autoclave-sterilizers.html.

 Siddiquee, Shafiquzzaman. “The Basic Concept of Microbiology.” Practical Handbook

of the Biology and Molecular Diversity of Trichoderma Species from Tropical Regions, 7

Sept. 2017, pp. 1–15, www.ncbi.nlm.nih.gov/pmc/articles/PMC7123386/, 10.1007/978-

3-319-64946-7_1.