RESEARCH 8

Quarter 3, Module 1
Topic 1: Microbiological Laboratory Techniques
Learning Objectives:
At the end of the module, you should be able to:
a. Provide an examples of the different laboratory techniques.
b. List down the laboratory procedures on sterilization, aseptic, inoculation and
incubation technique.
c. Identify the microbiological laboratory techniques and key information in growing
bacteria.
Read This!
Laboratory Techniques and Procedures
-Methods, procedures, and tests performed in the laboratory with an intended
application to the diagnosis of disease or understanding of physiological functioning.
The techniques include examination of microbiological, cytological, chemical, and
biochemical specimens, normal and pathological.
Explore!
Laboratory Techniques How it is done?
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Read This!

Laboratory techniques can be classified as microbiological, chemical and

physical. Below is an example of experiment on how to grow bacteria in a laboratory
setting, it explained the processes of different microbiological laboratory techniques that
comes up with the experiment.

EXPERIMENTS TO SHOW THE GROWTH OF BACTERIA - basic

techniques (similar techniques are used to grow fungi such as moulds and yeasts)
Microbiological media
Bacteria will grow on practically any source of organic food which
provides carbon compounds to be respired for energy, and nitrogen compounds to be
incorporated into proteins for growth. These substances are normally provided
dissolved in water. However, in nature, bacteria can break down solid and insoluble
substances by releasing enzymes into the substrate in which they are growing. These
substances are thus broken down or digested to simpler substances and the process is
called extracellular digestion because it takes place outside the bacterial cells.
 The two normal media used in bacteriology are a clear soup-like liquid nutrient
broth, usually in tubes, and nutrient agar, which is set into a jelly by the addition of a
seaweed extract called agar, and when melted poured into glass or plastic Petri dishes
- also known as "plates".

A standard carbon source is glucose, and nitrogen is often provided

by peptones (partially digested proteins), or inorganic salts. Minerals and vitamins
may also be provided, according to the growth requirements of the bacteria.
Combinations of chemicals (buffers) may be used to keep the pH stable. Measured
amounts of the concentrates are added to water, and dissolved to reconstitute the
media.

Sometimes, substances are mixed into media, in order to suppress growth of other
types of bacteria. There are many such selective media.

Microbiological Techniques
Sterilization, aseptic techniques, inoculation, incubation

These media must then be sterilized by heating in an autoclave (like a pressure

cooker) at 121°C (pressure 1 bar or 15 lb/sq. in.) for 15 minutes, which kills all living
organisms, including spores.

All apparatus used from this point onwards must be sterilised by heat (glassware -
160 °C for 2 hrs) or exposure to radiation.

Answer this!
Exercise no.1
Direction: List down inside each box the procedures in doing sterilization technique.

1. 2. 3.

4. 5.
 Aseptic techniques must be used to reduce the likelihood of bacterial
contamination. This usually involves disinfection of working areas, minimising possible
access by bacteria from the air to exposed media, and use of flames to kill bacteria
which might enter vessels as they are opened.

Answer this!
Exercise no.2
Direction: List down inside each box the procedures in doing aseptic technique.

1. 2. 3.

4. 5.

Bacteria may be introduced to the media (inoculated) by various means. Usually

the bacteria e.g. from a drop in a heat-sterilized loop are spread on the surface of
(ready set) agar. A similar technique is used with broth cultures.

Usually the bacteria e.g. from a drop in a heat-sterilized loop are spread on the
surface of (ready set) agar. A similar technique is used with broth cultures. Sometimes
bacteria in a liquid are introduced using a sterile pipette to the Petri dish before the
(fairly cool) agar medium is poured on top ("pour plates").
Answer this!
Exercise no.3
Direction: List down inside each box the procedures in doing inoculation technique.

1. 2. 3. 4.

8. 7. 6. 5.

9. 10.
 Then the Petri dishes containing agar or tubes containing broth are incubated, i.e.
put in a special apparatus at a fixed temperature (usually 37°C - human body
temperature, for possible pathogens - or 25°C for bacteria from the environment). In
schools, lower incubation temperatures are used in order to discourage the growth of
potential pathogens.

When growing bacteria, it is usual to invert the Petri dishes, so as to prevent

condensation droplets from falling onto the surface of the agar. Petri dishes are often
"sealed" at this stage to prevent people who handle them from contamination by
bacteria, which will multiply greatly. It is normal to use 2 strips of adhesive tape from
base to lid rather than attempt seal the circular edge of the Petri dish. This is to guard
against the possibility of anaerobic organisms growing due to lack of air. However, it
must be borne in mind that any drips from a partially sealed Petri dish are potential
sources of infection.

Results
Cultures are usually examined after 24 hrs. Incubation.

Liquid media such as broth become cloudy if bacteria are present. This could be
the result of only one bacterial cell originally entering the medium, then dividing
repeatedly to produce millions!

Bacteria on agar "plates" become visible as distinct circular colonies; each colony
should represent an individual bacterial cell (or group) which has divided repeatedly but,
being kept in one place, the resulting cells have accumulated to form a visible patch.

By an extension of this method using serial dilutions in sterilised liquids, the

number of bacteria in a given amount of sample, e.g. food, can be calculated.
After use, bacterial cultures, etc. must be sterilized by the use of heat, before disposal.
Answer this!
Exercise no.4
Direction: List down inside each box the procedures in doing incubation and growing
of bacteria technique.

1. 2. 3.

4. 5.

Assessment
1. Bacteria will grow on practically any source of organic food which provides
____?
a.Iron
b. Carbon
c. Petri dish
d. Oxygen
2. The clear soup-like liquid which uses as nutrient providing
normal media to grow bacteria used in bacteriology along with the nutrient
agar.
a. Nutrient broth
b. Plates
c. Agar
d. inoculation
3. What will be the color of liquid media such as broth if bacteria are
present?
a. Green
b. Red
c. Transparent
d. Cloudy
4. The common color of bacterial colonies in agar plates.
a. Whitish grey
b. Red/brown
c. Blue/green
d. White cream. Yellow
5. Technique done with petri dishes to prevent condensation when growing
bacteria.
a. Invert
b. Remove the lid
c. Heated
d. Incubated
6. Microbiological techniques which uses flame/heat to reduce the likelihood
of bacterial contamination to enter the vessel as it opened.
a. Inoculation
b. Aseptic technique
c. Sterilization
d. Incubation
7. A microbiological techniques performed by heating in an autoclave (like a
pressure cooker) at 121°C (pressure 1 bar or 15 lb/sq. in.) for 15 minutes,
which kills all living organisms, including spores.
a.Inoculation
 b.Aseptic technique
c.Sterilization
d.Incubation

8. The introduction of bacteria to a media by various means such as from a

drop in a heat-sterilized loop are spread on the surface of (ready set) agar
is called?
a.Inoculation
b. Aseptic technique
c.Sterilization
d. Incubation
9. Which is not required in inoculation?
a.Petri dish
b.Loop
c.Spectrophotometer
d.Flame/heat
10. A laboratory technique which was done by the use of special apparatus at
a fixed temperature.
a.Inoculation
b.Aseptic technique
c.Sterilization
d.Incubation

References:
Microbiological techniques - the basics (biotopics.co.uk)

www.definitions.net/definition/laboratory+techniques+and+procedures
 RESEARCH 8
Quarter 3, Module 2A
Topic 1: Chemical Laboratory Techniques
Learning Objectives:
At the end of the module, you should be able to:
11. List down the laboratory procedures of spectrophotometry.
12. Determine the protocols and techniques in doing spectrophotometry
analysis.

Read This!

Spectrophotometry
Spectrophotometry is a branch of electromagnetic
spectroscopy concerned with the quantitative
measurement of the reflection or transmission
properties of a material as a function of wavelength.
Spectrophotometry uses photometers, known as
spectrophotometers that can measure the intensity of a
light beam at different wavelengths.

spectrophotometer
Overview
Spectrophotometry quantifies the concentration of a specific substance that is
present in a sample by comparing the amount of light that goes into a sample versus
the amount of light that comes out of the sample at a specific wavelength.

The amazing thing about spectrophotometry is that it can theoretically be used to

measure any substance that absorbs light. For instance, spectrophotometry can
quantify nucleic acids, proteins and bacterial density, but it also can measure bitterness
compounds (IBUs, international bitterness units) in brewed beer!

Spectrophotometry primarily varies by the type of light being measured and the
wavelength selection of the instrument. First, either UV/Vis (ultraviolet/visible) light or
florescent light can be utilized in spectrophotometry.

A specific wavelength of UV/Vis light can be utilized to directly determine the

concentration of a substance, or the emission of fluorescent light can be used to
indirectly determine the concentration of a specific molecule.

Additionally, spectrophotometry can vary in regards to how wavelengths are

selected, either filter-based or monochromator. Filter-based instruments use optical
filters to select for the wavelength of interest and consequently filter out all other
wavelengths.

Monochromator instruments use diffraction gratings to select the desired

wavelengths. Both type of instruments have their benefits, filter-based have higher
sensitivity and lower detection limits, while monochromatic instruments have scanning
capability and wavelength flexibility.

Spectrophotometry is an experimental technique that is used to measure the

concentration of solutes in a specific solution by calculating the amount of light
absorbed by those solutes.This technique is powerful because certain compounds will
absorb different wavelengths of light at different intensities. By analyzing the light that
passes through the solution, you can identify particular dissolved substances in solution
and how concentrated those substances are. A spectrophotometer is the device used to
analyze solutions in a laboratory research setting.

Explore
Direction: With your idea about spectrophotometer, think of five titles in research and its
material/medium that will be measured by spectrophotometer.
Materials/Media Title of Research
1.
2.
3.
4.
5.

Read This!
PROCEDURES in SPECTROPHOTOMETER ANALYSIS
Part1
Preparing the Samples

1. Turn on the spectrophotometer. Most spectrophotometers need to warm up before

they can give an accurate reading. Turn on the machine and let it sit for at least 15
minutes before running any samples.
 13. Use the warm-up time to prepare your samples.

2. Clean the cuvettes or test tubes. If you are doing a lab for school, you may be
using disposable test tubes that don't need to be cleaned. If you are using cuvettes or
reusable test tubes, make sure they are properly cleaned before use. Rinse each
cuvette thoroughly with deionized water.

14. Take care with cuvettes as they can be quite expensive, particularly if they
are made from glass or quartz. Quartz cuvettes are designed for use in
UV-visible spectrophotometry.
15. When handling the cuvette, avoid touching the sides the light will pass
through (generally, the clear sides of the container).[2] If you accidently
touch these sides, wipe the cuvette down with a kimwipe (which are
formulated to prevent scratching the glass).

3. Load the proper volume of the sample into the cuvette. Some cuvettes have a
maximum volume of 1 milliliter (mL) while test tubes may have a maximum volume of 5
mL. As long as the laser producing the light is passing through the liquid and not an
empty part of the container, you will get an accurate reading.

16. If you are using a pipette to load your samples, use a new tip for each
sample to prevent cross-contamination.[3]

4. Prepare a control solution. Known as a blank, the control solution has only the
chemical solvent in which the solute to be analyzed is dissolved in. For example, if you
had salt dissolved in water, your blank would be just water. If you dye the water red, the
blank must also contain red water. The blank is the same volume as the solution to be
analyzed and kept in the same kind of container.

5. Wipe the outside of the cuvette. Before placing the cuvette into the
spectrophotometer you want to make sure it is as clean as possible to avoid
interference from dirt or dust particles. Using a lint free cloth, remove any water droplets
or dust that may be on the outside of the cuvette.[4]
Exercise no. 1
Direction: After reading the part 1 of spectrophotometry procedures, write your key-
takeaways (learnings) on each procedure.

1. Turn on the
spectrophotometer.

2. Clean the cuvettes

or test tubes.

3. Load the proper

volume of the sample
into the cuvette.

4. Prepare a control
solution.

5. Wipe the outside of

the cuvette.

Part 2
Running the Experiment

1. Choose and set the wavelength of light to analyze the sample with. Use a single
wavelength of light (monochromatic color) to make the testing more effective. The color
of the light chosen should be one known to be absorbed by one of the chemicals
thought to be in the test solute. Set the desired wavelength according to the
specifications of your spectrophotometer.
 17. In a classroom lab, the wavelength will likely be given to you.
18. Because the sample will reflect all light of the same color as it appears,
the experimental wavelength will always be a different color than that of
the sample.
19. Objects appear as certain colors because they reflect light of particular
wavelengths and absorb all other colors. Grass is green because the
chlorophyll in it reflects green light and absorbs everything else.

2. Calibrate the machine with the blank. Place the blank into the cuvette holder and
shut the lid. On an analog spectrophotometer, there will be a screen with a needle that
moves based on the intensity of light detection. When the blank is in, you should see
the needle move to the right. Record this value in case you need it for later. With the
blank still in the machine, move the needle to zero using the adjustment knob.
20. Digital spectrophotometers can be calibrated in the same way, they will
just have a digital readout. Set the blank to 0 using the adjustment knobs.
21. When you remove the blank, the calibration will still be in place. When
measuring the rest of your samples, the absorbance from the blank will
automatically be subtracted out.
22. Be sure to use a single blank per session so that each sample is
calibrated to the same blank. For instance, if you blank the
spectrophotometer, then analyze only some of samples and blank it
again, the remaining samples would be inaccurate. You would need to
start over.
3. Remove the blank and test the calibration. With the blank removed the needle
should stay at 0 (zero) or the digital readout should continue to read 0. Place the blank
back into the machine and ensure the needle or readout doesn't change. If the machine
is properly calibrated with your blank, everything should stay at 0.
23. If the needle or readout is not 0, repeat the calibration steps with the
blank.
24. If you continue to have problems, seek assistance or have the machine
looked at for maintenance.

4. Measure the absorbance of your experimental sample. Remove the blank and
place the experimental sample into the machine. Slide the cuvette into the designated
groove and ensure it stands upright. Wait about 10 seconds until the needle is steady or
until the digital numbers stop changing. Record the values of % transmittance and/or
absorbance.
25. The absorbance is also known as the optical density (OD).
26. The more light that is transmitted, the less light the sample absorbs.
Generally, you want to record the absorbance values which will usually be
given as a decimal, for example, 0.43.
27. If you get an outlying result (such as 0.900 when the rest are around
0.400), dilute the sample and measure the absorbance again.
28. Repeat the reading for each individual sample at least 3 times and
average them together. This ensures a more accurate readout.
5. Repeat the test with successive wavelengths of light. Your sample may have
multiple unknown compounds that will vary in their absorbance depending on
wavelength. To eliminate uncertainty, repeat your readings at 25 nm intervals across
the spectrum. This will allow you to detect other chemicals suspected to be in the
solute.

Exercise no. 2
Direction: After reading the part 2 of spectrophotometry procedures, write your key-
takeaways (learnings) on each procedure.

1. Choose and set the

wavelength of light to
analyze the sample with.

2. Calibrate the machine

with the blank.

3. Remove the blank and

test the calibration.

4. Measure the
absorbance of your
experimental sample.

5. Repeat the test with

successive wavelengths of
light.

Part 3
Analyzing the Absorbance Data

1. Calculate the transmittance and absorbance of the sample. Transmittance is how

much of the light that passed through the sample reached the spectrophotometer.
Absorbance is how much of the light has been absorbed by one of the chemicals in the
solute. Many modern spectrophotometers have an output of transmittance and
absorbance, but if you recorded intensity, you can calculate these values.[5]
29. The transmittance (T) is found by dividing the intensity of the light that
passed through the sample solution with the amount that passed through
the blank. It is normally expressed as a decimal or percentage. T =
I/I0 where I is the intensity of the sample and I0 is the intensity of the blank.
30. The absorbance (A) is expressed as the negative of the base-10 logarithm
(exponent) of the transmittance value: A = -log 10T.[6] For a T value of 0.1,
the value of A is 1 (0.1 is 10 to the -1 power), meaning 10% of the light is
transmitted and 90% is absorbed. For a T value of 0.01, the value of A is
2 (0.01 is 10 to the -2 power), meaning 1% of the light is transmitted.

2. Plot the absorbance values versus the wavelengths on a graph. The

absorbance value is plotted on the vertical y-axis against the wavelength of light used
for a given test plotted on the horizontal x-axis. Plotting the maximum absorbance
values for each wavelength of light tested, produces the sample's absorbance spectrum
and identifies the compounds making up the test substance and their proportions.
31. An absorbance spectrum usually has peaks at certain wavelengths that
can allow you to identify specific compounds.

3. Compare your absorbance spectrum plot to known plots of specific

compounds. Compounds have unique absorbance spectrum and will always produce
a peak at the same wavelength every time they are measured. By comparing your plots
of unknown compounds to those of known compounds, you can identify the solutes that
compose your solution.
32. You can also use this method to identify contaminants in your sample. If
you are expecting 1 clear peak at a specific wavelength and you get 2
peaks at separate wavelengths, you know something is not right in your
sample.

Exercise no. 3
Direction: After reading the part 3 of spectrophotometry procedures, write your key-
takeaways (learnings) on each procedure.

1. Calculate the
transmittance and
absorbance of the
sample.

2. Plot the
absorbance values
versus the
wavelengths on a
graph.

3. Compare your
absorbance spectrum
plot to known plots
of specific
compounds.

Assessment
Direction: Write the letter of the correct answer.

1. Spectrophotometers need to warm up for how many minutes?

a. 10minutes
b. 2 minutes
c. 15 minutes
d. 30 minutes
2. How properly clean cuvette before use?
a. Rinse each thoroughly with deionized water.
b. Wash with soap and water
c. Wash with water only
d. Gently wipe with dry cloth.
3. What you should do when accidentally touched the cuvette’s side where the
light pass through?
a. scrape with scalpel
b. wipe with hand
c. wipe the cuvette down with a kimwipe
d. clean with a brush
4. Laser producing light of spectrophotometry should pass the_________?
a. Empty part of cuvette
b. Liquid filled cuvette
c. Between the liquid and empty part of cuvette
d. None of the above
5. Before placing the cuvette into the spectrophotometer.
a. Shake for 3 minutes
b. Wipe the outside part to free from dust or droplets.
 c. Clean with mild soap and water
d. Just place as it is
6. To make the testing more effective
a. Use multichromatic color
b. Use monochromatic color
c. Disregard colors reflected by the sample
d. Wavelength has nothing to do with the experiment
7. To have an accurate reading of the spectrophotometer, what is the proper
technique?
a. Use multiple blank per session
b. Replace the first blank
c. Test different sample with multiple blank
d. Use only single blank per session
8. In calibrating spectrophotometer
a. Needle must stay zero when the blank remove
b. Needle must not zero
c. After placing back the blank, needle should change reading
d. Recalibrate if needle is always zero
9. Record the value percent of transmittance and absorbance after placing the
sample for how many seconds?
a. 1 seconds
b. 4 seconds
c. 15 seconds
d. 30 seconds
10. To get an accurate reading of the absorbance.
a. Repeat the reading for each sample 3 times then get the average.
b. Repeat the reading for each sample 5 times then get the average
c. Repeat the reading for each sample 6 times the get the average
d. Read the sample for only once

References:
The Importance of Flexibility in Spectrophotometry (news-medical.net)
How to Do Spectrophotometric Analysis: 13 Steps (with Pictures) (wikihow.com)
https://en.wikipedia.org/wiki/Spectrophotometry