MARMARA UNIVERSITY

FACULTY OF ENGINEERING
DEPARTMENT OF BIOENGINEERING

2024-2025 FALL SEMESTER

BIOE 2201.1
MICROBIOLOGY FOR BIOENGINEERS
LABORATORY MANUAL

INSTRUCTOR: Prof. Ahu ALTINKUT UNCUOĞLU

RESEARCH ASSISTANTS: Tuğba ÖZGÖREN CAN, MSc

CONTENTS

How to write laboratory reports? i

Safety rules and guidelines v

Experiment 1: Preparation of Culture Media and Sterilization 1

Experiment 2: Pure Culture (Single Colonies) Isolation Methods 8

Experiment 3: Quantitative Determination of Bacterial Numbers 13

Experiment 4: Effects of Temperature, pH, Osmotic Pressure and Non-ionizing

Radiation on Microbial Growth 16
Experiment 5: Staining Bacteria 20

Experiment 6: Screening of E. coli and B. subtilis Cells for Antibiotic and Heavy
Metal Resistance by Paper Disc-Agar Diffusion Assay 23
 THINGS YOU NEED TO KNOW BEFORE BEGIN DOING THE EXPERIMENTS:

HOW TO WRITE LABORATORY REPORTS

 The soft copy of your reports should be submitted to turnitin.com (Plagiarism system) untill the
deadline announced by the lab assistant for each experiment.
 To be able to submit your reports, you should sign in turnitin.com as student and register to the
course “MICROBIOLOGY FOR BIOENGINEERS 2024”
CLASS ID: 45834122
ENROLLMENT KEY: 2024BIOE2201

 If you can not submit your report to the system on schedule, you will be failed from that report and
your grade will be zero.

 Similarity in turnitin should be less than 20%. If you upload your reports to the system earlier, you
can see the similarity rate. So you can make your corrections and upload them again.

 The average grades of your lab reports will constitute 30% of your final grade.

 25-point question will come from the laboratory section in your final and midterm exams.

 You will fail the course if you do not participate more than 1 experiment!

 Reports will be written by the groups created by you. Only the discussion part will be written
individually. All discussion texts will be added to the end of the report.

 Students who do not participate in the experiment will not be able to write the report of that
experiment.

 Repeat students are obliged to write reports, too.

 Report files should be entitled as: BIOE2001_Exp#_Name Surname1_Name Surname2..

 Laboratory reports will be written in English.

 A report should always be orderly and neat.

 The language should be consistent throughout the report. Third-person singular passive voice is
the acceptable language for the reports.

i
 No hand written reports (Please use Word etc.)

 No freehand graphs, tables (Please use Excel etc.)

Writing style and structure

• Plain text font should be Times New Roman in 12 pt size.

• Bold letters are generally used in headings. However, if you want to draw attention to some
words or sentences, these can be written with bold or italic form of the letters.

• Plain texts should be written with 1.5 line spacing and space before each paragraph should be
added.

• All titles should be aligned to the left but plain texts should be justified.

• No title is written as the last line of the page. At least 2 lines of plain text should be positioned.
If it is not possible, the title starts from the next page.

ii
REPORT SECTIONS:

 Cover Page consists of the course code and the course title, Marmara University logo, the number
and the title of the experiment, the name of the student, the date of experiment, the date of
submission of the report, to whom it is submitted.

 The introduction gives the aim and relevance of the experiments. It should make the reading and
understanding of the report easier. It should not be too long (maximum three pages) and it should
cover the following topics: What are the important parts of this experiment? What subject did you
investigate? You can give some brief info about the tools used during the experiment i.e. chemicals,
equipment used. Indicate the reference of each information given. Please give the name and the
reference number of the figures, tables, pictures used. DO NOT DIRECTLY COPY AND PASTE FROM
THE WEB SITES AND FROM OTHER STUDENTS.

 Materials and Methods part describes all materials (e.g. chemicals, organisms, solutions, buffers,
equipment pieces) that were used for the experiment. On the other hand, it includes a detailed
description of the experiment method. Enough details should be given so that someone who reads
your reports should be able to perform that experiment exactly as you did. Laboratory manuals
give us only general instructions. Sometimes, we may modify the method considering the current
opportunities in our laboratory. Thus, please do not copy from the lab manual, write the detailed
method you follow in the lab with your own words.

 Results part includes the results and observations obtained in the experiments. Generally, the best
way to show results is to use graphs or tables at suitable parts of reports. For microscopy, the
drawings of your observations also belong to this section of reports. Using colored pencils makes
your drawings more understandable. On the other hand, you should indicate the important parts
with arrows and name them. All images, graphs, tables should be numbered and tittled. Numerical
values should be given with their units.

 Discussion includes a detailed evaluation of the results obtained. The important findings and the
sources of errors should be discussed. Suggestions, in order to overcome these errors and improve
the experiments, could be made. On the other hand, the results should be compared with the
information in the literature.

 References should be sources that you used in the report preparation. You should list, the full name
of the book/paper, names of authors, year of publishing, number of the editions, the publishing
company etc. The retrieved dates of web sites should be given. Please do not use the laboratory

iii
manual as a reference in your reports and please try to use different references from the laboratory
manual. Additionally, please try to use up to date papers and books instead of web sites. If you have
to use web sites as reference, you should use websites from universities, governmental
organizations and other formal sources.

Example for the book reference:

Pollock, H.M., Smith, D.A., 2002. The use of near-field probes for vibrational spectroscopy and
photothermal imaging, in Handbook of vibrational spectroscopy, J.M. Chalmers and P.R. Griffiths
(eds), John Wiley & Sons Ltd, Vol. 2, pp. 1472 - 1492.

Example for the paper reference:

Gustafsson, M. G. L., 2000. Surpassing the lateral resolution limit by a factor of two using structured
illumination microscopy. J. of Microsc. 198(2), 82–87.

Example for the web site reference:

Abramowitz, M., Davidson, M.W. 22 Feb 2007. "Introduction to Microscopy", Molecular

Expressions. (http://micro.magnet.fsu.edu/primer/anatomy/introduction.html).

iv
 SAFETY RULES AND GUIDELINES

1. Do not eat, drink or smoke in the laboratory. Especially, no chewing gum!

2. No cell phone in the lab!

3. Please wear a lab coat in the lab. Otherwise, you will not be allowed to do the experiment.
Using gloves during experiments is mandatory for your health. Please use additional
protection equipment if necessary.

4. Do not wear a lab coat outside the lab area.

5. Do not mouth pipet.

6. If you have a wound, an open cut or a burn, please protect it with a bandage.

7. Clean all working area with technical alcohol (70 %).

8. Discard material only where indicated.

9. If you spill or break something, please inform your instructor immediately.

10. Please return all common materials used by others to appropriate place, as soon as you have
finished using it. Please organize all the trashes from your experiment.

11. Be careful with open flames and flammable material etc.

12. Wash your hands after every laboratory exercise.

13. BEFORE YOU LEAVE THE LABORATORY CHECK THE FOLLOWING:

 Is your equipment (e.g. microscope, heater etc.) safely turned off?

 Have you turned the GAS and WATER FLOW OFF?

 Have you brought back the COMMON MATERIAL you had used?

 Have you CLEANED YOUR BENCH?

v
 Experiment 1: Preparation of Culture Media and Sterilization
Introduction:

1. Sterilization

Physical or chemical process that serves to reduce the number of microorganisms on any
inanimate object is safe for subsequent handling is called decontamination. Different
methods applied for the control of microorganisms in term of decontamination is
described below;

Sanitization: Reduction of microbial populations to levels considered safe by public

health standards, such as those applied in restaurants.

Disinfection: Elimination of most or all pathogens (except bacterial spores) from nonliving
objects by pasteurization or liquid chemicals (disinfectants) such as alcohols, chlorine
compounds, phenolics etc.

Pasteurization: A method of disinfecting liquids in which hot water at temperatures

less than 100°C is used. Today it is preferred to eliminate pathogens from milk and most
other beverages.

Antisepsis: Removal of pathogens from skin or mucous membranes

Sterilization: Microorganisms are abundant in our environment. They are present in air,
on human body, on every surface and object etc. Therefore, all necessary materials have
to be sterilized before use in order to prevent contamination during a microbiological
study. Sterilization completely destroys microorganisms, including bacterial endospores.
Sterilization could be achieved by various methods. These methods include the utilization
of high-pressure steam (autoclave), dry heat (oven), chemical sterilants (glutaraldehydes
or formaldehyde solutions) or physical agents (radiation).

1.1.Heat Sterilization

1.1.1. Red Heat (Flaming)

This kind of sterilization is used for some equipments that are not damaged by heat such
as wires, loops, metal instruments etc. These instruments are sterilized under Bunsen
flame until they turn glowing dred.

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1.1.2. Flaming After Ethanol Dipping
This method is frequently used for scalpels, spatules etc. but it does not necessarily
achieve a sterilization.

1.1.3. Dry Heat (Hot air)

This is applied in electrically heated ovens (dry-heat sterilizers) that are thermostatically
controlled and it is equipped with a large circulating fan in order to have a constant
temperature at every point in the oven. Dry-heat sterilization requires use of higher
temperatures, so it can be used only with glass or metal objects (Petri dishes, flasks,
pipettes etc.). It generally melts other substances.
1.1.4. Boiling in Water
Boiling for 5-10 min is sufficient to kill non-sporing organisms, but many bacterial spores
will survive. This method is useful where sterility is not essential (as is the case with many
selective media) or where better methods are not available or are unsuitable.

1.1.5. Koch’s Steam Sterilizer

Sterilization in a Koch’s steam sterilizer (steamer) in steam at atmospheric pressure and
approximately 100oC is used for media etc. that are damaged when exposure to
temperatures above 100 oC (sugar etc.) and milk.

1.1.6. Autoclave
Autoclaving is the most efficient method of sterilizing culture media that should be used
for all media capable of withstanding the high temperature. It is also used for glassware
and instruments and for sterilizing cultures and contaminated material before washing.
Proteins of bacteria are denatured by steam and this is the reason of death of
microorganisms. There is a direct relationship between the gas pressure and temperature.
If the pressure of the gas increases, the temperature increases as well. The actual
temperature inside the autoclave depends on the steam pressure. The temperature of
saturated steam at atmospheric pressure is approximately 100°C. Sterilization in the
autoclave is usually achieved by autoclaving at 121°C for 15-30 min in pure saturated
steam at 103.4 kPa (15 lb in-2) above atmospheric pressure.

1.2.Gas Sterilization
Heat-liable materials like disposable syringes, petri dishes culture tubes, plastic bags,
plastic filtration devices and various plastic ware materials could be used in gaseous

2
autoclave. Ethylene oxide (ETO) and formaldehyde gas are known gases used in gaseous
autoclave.

1.3.Radiation
Ultraviolet (UV) light has been used to help disinfect the air for more than 50 years. UV
irradiation has very limited energy and does not penetrate dust, mucous or water. It is
generally used for surface sterilization of glassware materials. Some materials like
pharmaceuticals are sterilized by radiation for “cold sterilization”. Gamma rays from
cobalt-60 or cesium-139 source are generally used for this type of sterilization.

1.4. Filtration
Microorganisms may be removed from liquids by passing them through filters with very
small pores that trap bacteria. However, filtration is not suitable for mycoplasmas or
viruses. The method is used for sterilizing serum for laboratory use, antibiotic solutions
and special culture media that would be damaged by heat.

2. Preparation of Culture Media

Examination of the formula of many culture media designates a few key nutrient
ingredients, and several other components added for the selection and/or identification
of separate groups or species of microorganisms. Culture media can be liquid or solid.
2.1. Media Types
2.1.1 Synthetic Medium
A synthetic, or chemically defined, medium contains known amounts of pure chemical
substances.
2.1.2 Complex Medium
A complex, or non-synthetic, medium is chemically undefined medium that contains
complex materials that are rich in nutrients and vitamins (e.g. yeast extract, peptone).

2.1.2.1. Broth Medium (Liquid)

Liquid medium is a suitable way to provide available environment for bacterial growth in
a test tube or a flask. There are various recipes of broth medium for different types of
microorganisms.
2.1.2.2. Solid Medium
Gelatin or agar is generally used as a solidifying agent in liquid media. Agar, which is
extracted from marine red alga, is usually preferred because; few microorganisms can

3
degrade agar; it is transparent, it solidifies at about 42°C; once it solidifies it takes elevated
temperatures as high as 85 oC to become liquid again. Agar is typically used in a final
concentration of 1-2% for solidifying culture media.
Luria-Bertani (LB) is a common medium used for bacteria. Composition of LB Broth and LB
Agar are shown in Tables 1.1 and 1.2, respectively.

Table 1.1: Components of LB Broth

Components Amount (For 1 Liter)

Tryptone 10 g
Yeast Extract 5g
NaCl 10 g

Table 1.2: Components of LB Agar

Components Amount (For 1 Liter)
Tryptone 10 g
Yeast Extract 5g
NaCl 10 g
Agar 15 g

2.1.2.3. Selective Medium

Selective medium is formulated in a way that it supports the growth of a specific organism
and restrain the others.

2.1.2.4. Differential Medium

It helps distinguish microorganisms from one another based on a growth characteristic,
which typically induce a visible change.

2.1.2.5. Enrichment Medium

Enrichment medium is designed to provide essential nutrients for the growth of a specific
microorganism (e.g. Salmonella sp.).

Materials:
 Tryptone
 Yeast extract
 NaCl
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  Agar
 Aluminium foil
 Bunsen burner
 Petri dishes
 Graduated cylinder
 Flasks
 Cotton swabs
 Tubes

Methods:
A) Preparation of LB broth
1) Weigh the necessary ingredients for the preparation of 250 mL LB broth according to
the recipe outlined in Table 1.1.

2) Place the ingredients into a clean 250 mL graduated cylinder and add 100 mL
deionized water. Stir until all dissolve completely.

3) Make solution volume up to 250 mL by adding more deionized water and stir for
homogeneity.

4) Distribute 200 mL of LB broth into clean 500 mL Erlenmeyer flasks so that each
contains 100 mL LB broth and fit loose cotton plugs to the flasks. Place them in the
autoclave.

5) Prepare 10 glass tubes in a test-tube rack and pipette 5 mL of LB broth into each.
Loosely cap the tubes and place them into the autoclave.

6) For sterilization;

a. Switch the autoclave on and add the required amout of distilled water as
directed in user manual.

b. Loosely cap the tubes, apply autoclave tapes on each tubes and place them into
the autoclave chamber.

c. Close the lid tight, the light indicating that lid is open should be off.

d. Adjust the program for sterilization at 121 oC and 1.0 atm for 15 min.

e. After sterilization is over, the device will give a finish warning. Open the lid
carefully and let the media cool down to a safe temperature.

f. Wear autoclave gloves, take the tubes out and tighten the caps.

5
B) Preparation of LB agar plates & agar slants
1) Prepare 250 mL of LB agar according to the recipe given in Table 1.2

2) Transfer LB agar into a 500 mL Erlenmeyer flask and place a loose cotton plug to seal
the flask.

3) Sterilize agar solution at 121 oC and 1.0 atm for 15 min. Let it cool down to 50-55 oC.

4) Wipe the benchtop with 70% Ethanol solution. Remove sterile petri plates from
plastic bag. Light the Bunsen burner. Place the plates around the burner on the
bench.

5) Flame the neck of the flask containing LB agar medium, pour a thin layer of LB agar
into each plate, swirl each plate in a circular motion to make agar cover the bottom
completely and to remove any bubbles.

6) Let plates stand their lids partly-open until agar solifies. As soon as it becomes solid,
close the lids entirely, place the plates into a plastic bag in an inverted position and
preserve at 4 oC until use.

7) Spare 20 mL of sterile LB agar for agar slant preparation.

8) Prepare 4 pre-sterilized glass tubes with caps in a tube rack.

9) Pipette 5 ml of sterile LB agar into each tube aseptically and close the caps loosely.

10) Tilt the tubes onto a solid surface at an approximate angle of 45o and hold them still
in that position until agar solidifies having a slanted surface.

11) As soon as agar solidifies, tighten the caps and store the slants at 4 oC.

References

 Collins, C.H., Lyne, M.P., Grange, J.M. 1989. Microbiological Methods, 6th Edition,
Butterworth &Co Ltd.

 Engelkirk, P. G., Duben-Engelkirk, J., & Fader, R. C. 2020. Burton's microbiology for the
health sciences, 11th Edition, Jones & Bartlett Publishers.

 Harrigan, W. F., 1998. Laboratory methods in food microbiology, 3rd Edition, 17-23,
Academic Press, California.

 Morris, E.J., 1972. The practical use of ultraviolet radiation for disinfection purposes.
Med Lab Technol 29(1): 41–47.

 Reichert, M., & Young, J. H. 1997. Sterilization technology for the health care facility,
2nd Edition, Jones & Bartlett Learning.

6
 Seely, H.W., Vandemart, J.P., Lee, J.J. 1991. Microbes in Action, 4th Edition, W.H.
Freeman and Company.

 Stainer, Y.R., Doudoroff, M., Adelberg, E.A. 1963. The Microbial World, 2nd Edition,
Prentice-Hall, Inc.

 Prescot, H. 2002. Laboratory Exercises in Microbiology, 5th Edition, McGraw-Hill

Companies.

7
 Experiment 2: Pure Culture (Single Colonies) Isolation Methods

Introduction:
1) Pure Culture
Samples and specimens for microbiological examination may contain mixtures of many
different microorganisms. On the other hand, a pure culture contains only a single kind of
microorganism that has originated from a single cell. Pure cultures of microorganisms are
preferred in experimental studies including the determination of growth characteristics,
pathogenity, metabolism, and antibiotic susceptibility, etc.

2) Isolation of Fungi by Brefeld

Oscar Brefeld reported the growth of fungal colonies from single spores on gelatin in 1872.
Gelatin has been used in culture for the isolation of fungi since then. However, some bacteria
have ability to degrade gelatin, which is a protein derived from animal tissue. This destroys
the growth surface on the petri plate making it unsuitable as a bacterial growth medium.
Therefore, gelatin usage is generally successful for the isolation of fungi, not for bacteria.

3) Isolation of Bacteria: Dilution Technique

If a mixture of bacteria is serially diluted (Figure 2.1) with a sterile medium, it is possible to
isolate a single culture. In history, scientists tried potato, bread, egg albumin etc. in order to
isolate bacterial species because it was easier to isolate them when they were grown on solid
surfaces. Agar is a suitable chemical for solidifying bacterial media. Nutrient medium solidified
by agar on petri dishes is typically used for the isolation of bacteria.

8
 Figure 2.1: Serial Dilution

Serial dilution is employed in the application of each of the following techniques to isolate
bacteria. These techniques are pour plate, streaking and spreading. They depend on the
separation of aggregates of bacteria so that each will grow into a separate colony. Colonies
resulting from these manipulations may then be subcultured for further examination.

Pour plate (Figure 2.2) is made by adding a diluted culture from a serial dilution into melted
agar medium. After medium is mixed, it is poured into an empty petri dish. As soon as agar
medium cools, and solidifies petri plate is placed into the incubator and kept until colonies
develop both within the medium and on its surface. Sometimes, cells suspended in melted
agar may get damaged by the heat of agar medium during preparation, and then they do not
form colonies. Additionally, cells at the subsurface of agar medium may be organised in
smaller colonies. Spread plate technique eliminates such problems. In the spread plate
technique (Figure 2.2), spreaders are used. Diluted sample is spread on the solid media with
the help of this rod. After incubation, the formation of colonies becomes visiable on the
surface of solid medium plate.

9
 Figure 2.2: Spread Plate Technique and Pour Plate Technique

In streaking technique (Figure 2.3), an inoculating loop is used. Bacterial colonies are picked
up on a sterile loop and this loop is moved lightly along the agar medium surface, depositing
streaks of bacteria on surface. The inoculating loop is flamed and a few bacteria are picked up
from the region already streaked. These movements are repeated 3 or 4 times on the non-
streaked area of solid medium.
Material:
 A bacterial culture (E. coli)
 Nutrient broth (solid and liquid)
 Sterile saline solution (0.9% NaCl)
 Bunsen burner
 Incubator
 Micropipette and its tips
 Petri dishes Figure 2.3: Streaking Technique
 Inoculation loop
 Glass/disposable cell spreader
 Test tubes with caps
 Beaker
 Ethanol (technical grade)

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Methods:
A) Streaking Technique
1. Label the bottom of the plate.

2. Sterilize a platinum wire loop by flaming until it turns red. Cool it in air.

3. Used cooled loop to pick up bacteria. Touch the loop to a single, well-isolated bacterial
colony growing on the surface of solid medium.

4. Open the lid of petri dish.

5. Streak the bacteria on the loop onto the plate containing agar medium. Sterilize the
loop by flaming and cool it by stabbing into a region of the agar medium that is free of
bacterial cells. Pass the loop across one end of primary steak and spread bacteria on
the loop into a fresh region of the agar medium.

6. Sterilize and cool the loop again and streak from one end of a secondary streak.

7. Repeat previous step one more time.

8. Replace the lid on the plate.

9. Incubate it in an inverted position at 37oC for 24 to 48 hours.

10. After incubation, well-separated colonies should be visible in the area of final streak.

B) Spreading Technique
1. Label the bottoms of agar plates suitably (e.g. species’ name, dilution factor, date).

2. Mix the E. coli culture tube thoroughly and pipette 40 µl of culture onto the center of
a previously prepared agar plate.

3. Dip the L-shaped glass spreader into a beaker of ethanol and then tap it on the side of
the beaker to remove any excess ethanol. Pass the ethanol-soaked spreader through
the flame to burn off all the alcohol, and allow it to cool inside the lid of a sterile petri
plate (Figure 2.4 below).

4. Spread the sample evenly over the agar surface with the sterilized spreader.

5. Prepare more replicate plates as described above.

6. Incubate the plates at 37°C for 24 to 48 hours in an inverted position.

7. Examine the spread plates for apparent colonies and record your results in your report.

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 Figure 2.4: Sterilization of the spreader.

CAUTION!
Do not place the beaker of ethanol near the flaming Bunsen burner. Do not return the hot
glass rod to the beaker of ethanol!

References:
 Prescot, H. 2002. Laboratory Exercises in Microbiology, 5th Edition, McGraw-Hill
Companies.

 Black, J.G. 2002. Microbiology, Principles and Explorations, 5th Edition, John Wiley and
Sons, Inc.

 Collins, C.H., Lyne, M.P., Grange, J.M. 1989. Microbiological Methods, 6th Edition,
Butterworth &Co Ltd.

 Johnson, T.R., Case, C.L. 2001. Laboratory Experiments in Microbiology, 6th edition,
addison Wesley Longman, Inc. California.

 Stainer, Y.R., Doudoroff, M., Adelberg, E.A. 1963. The Microbial World, 2nd Edition,
Prentice-Hall, Inc.

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 Experiment 3: Quantitative Determination of Bacterial Numbers
Introduction:
There are various methods applied for measuring bacterial growth quantitatively. Standard
plate counts, direct microscopic counts and turbidimetric analysis are the most widely used
methods of growth measurement. Other methods include most probable number (MPN)
method, filtration, metabolic product measurement, cell dry weight determination etc.

Standard Plate Count: Principle of this method relies on the fact that a microbial colony that
appears on the surface of an agar plate is originated from a single viable microorganism.
Theoretically, each single cell divides several times during incubation and forms a visible
“colony” on agar surface. Microbial number is reported in terms of CFU, which stands for
“colony forming unit”. There are lower and upper limits for counting bacterial number in this
method. Plates having colonies higher than 300 or less than 30 are not acceptable for
statistical reasons. Serial dilutions of the microbial culture are prepared in order to obtain
satisfactory colony numbers. After preparing serial dilutions, a specific amount of each
dilution (typically 100 µl) is spread on an agar plate. At the end of the incubation period
colonies appeared on the agar surface are counted. Multiplying counted colony numbers by
the dilution factor and dividing by the culture volume used to inoculate the agar plate,
microbial number in main culture can be computed in CFUs per milliliter.

Direct Microscopic Counts: Direct microscopic counts can also be done in order to determine
microbial numbers in a liquid culture. Various equipment can be used for that purpose. Among
those, hemocytometers are special “glass slides” with calibrated areas of known size.

Turbidimetric Analysis: Increase in the turbidity of a culture is an indication of microbial

growth. When turbidity is measured with a photometric device like a spectrophotometer or
colorimeter, the evaluation of growth can be estimated proportionally because the light
transmitted decreases as cell population increases. However, turbidity of a culture does not
show that all cells in the culture are alive.

13
Materials:
 Overnight E. coli culture
 Petri plates containing solid complex medium
 Liquid complex medium
 Sterile saline solution (0.9% NaCl)
 Test tubes
 Micropipettes and tips
 Vortex mixer
 Bunsen burner
 Glass/disposable cell spreader
 Beaker

Methods:
A) Standard Plate Count
1. Label the bottom of agar plates suitably (e.g. species’ name, dilution factor, date).

2. Label 7 pre-sterilized microcentrifuge tubes and deliver 900 µl of sterile saline solution
into each one.

3. Mix the E. coli culture tube thoroughly, withdraw 100 µl from the tube aseptically and
transfer it to the saline tube 1. This represents a 10–1 dilution.

4. Mix the contents of saline tube 1 thoroughly and using aseptic technique, immediately
inoculate saline tube 2 with 100 µl from saline tube 1; a 10–2 dilution.

5. Carry on preparing serial 10-fold dilutions as instructed until you have 10-7 dilution of
E. coli culture in the 7th microcentrifuge tube.

6. Mix the contents of 10-3 dilution tube and pipette 100 µl of the diluted culture onto
the center of a previously prepared agar plate.

7. Spread the sample evenly over the agar surface with the sterilized spreader.

8. Prepare two more replicate plates for 10-3 dilution.

9. Repeat steps 6 to 8 for the rest of the bacterial dilutions so as to have 3 inoculated agar
plates for each dilution.

10. Incubate the plates at 37°C for 24 to 48 hours in an inverted position.

11. At the end of the incubation period, select all of the petri plates containing between
30 and 300 colonies. Count the colonies on each plate.

14
 12. Calculate the cell number (CFU) per ml of the overnight E. coli culture.

B) Turbidimetric Determination of Bacterial Numbers

1. Prepare 6 sterile test tubes in a test-tube rack and label them with numbers starting
from 1. These tubes will be used for the serial dilution of overnight E. coli culture.

2. Add 2.0 ml of sterile liquid broth aseptically into each tube.

3. Mix E. coli culture tube thoroughly, withdraw 2.0 ml from the tube aseptically and
transfer it to the test tube 1; a 1/2 dilution.

4. Mix the 1/2 dilution tube completely, then pipette 2.0 ml from its contents and deliver
to the test tube 2, a 1/4 dilution.

5. Continue diluting consecutively until you have 1/64 dilution of E. coli culture in test
tube 6.

6. Turn on the spectrophotometer, set the wavelength to 600 nm.

7. Pipette 1.0 ml of sterile broth into a clean spectrophotometer cuvette, make sure that
there is no bubble in the solution and no impurity on the outside walls especially where
light passes through. Then, place it into cuvette holder in the device, and close the lid.
Press “blank/zero” button and wait until 0.00 absorbance is read on the screen.
Remove the cuvette.

8. Mix the contents of test tube 1 (1/2 dilution) thoroughly and deliver 1.0 ml to a clean
cuvette. Place it into the spectrophotometer, close the lid and press “Read” button.
Wait until an absorbance value appears on the screen. Write down the dilution ratio
and corresponding absorbance value at 600 nm.

9. Measure absorbance of the rest of dilutions as described above and record the values.

10. Calculate the cell number in each dilution by taking into account the calculated cell
number in overnight E. coli culture (found in part A).

11. Plot cell number vs absorbance values at 600 nm. Comment on your graph.

References:
 Prescot, H. 2002. Laboratory Exercises in Microbiology, 5th Edition, McGraw-Hill
Companies.
 Black, J.G. 2002. Microbiology, Principles and Explorations, 5th edition, John Wiley and
Sons, Inc.
 Collins, C.H., Lyne, M.P., Grange, J.M. 1989. Microbiological Methods, 6th Edition,
Butterworth &Co Ltd.
 Stainer, Y.R., Doudoroff, M., Adelberg, E.A. 1963. The Microbial World, 2nd Edition,
Prentice-Hall, Inc.

15
 Experiment 4:
Effects of Temperature, pH, Osmotic Pressure and Non-ionizing Radiation
on Microbial Growth
Introduction:
1. Temperature
Temperature, pressure and radiation are the physical factors that affect bacterial growth and
death. When microorganisms are exposed to temperatures higher than their optimal
temperature for growth, only resistant spores may survive. As a consequence of biochemical
changes inside the cell and loss of water due to heat, cell death occurs. Generally,
temperatures lower than the growth temperature of microorganisms may not result in their
death.
Microbial growth can be controlled by controlling temperature through several methods
including, exposing to direct flame, or dry heat (hot air oven) (for example; 160oC in 2 hours
for bacterial spores), boiling water (water molecules conduct heat better than air molecules),
pasteurization (to decrease the number of bacteria in milk and destroy microorganisms that
cause food spoilage and human diseases) and most frequently sterilizing in an autoclave.

2. Radiation
Electromagnetic radiation can be differentiated according to its wavelength, frequency and/or
energy. As the wavelength of radiation decreases energy emitted by the radiation increases.
There are two forms of electromagnetic radiation; ionizing radiation and non-ionizing
radiation. Ionizing radiation such as x-rays or gamma radiation carries enough energy to
remove electrons from molecules in a cell. After electrons are removed from molecules, ions
called free radicals are formed. Free radicals can damage molecules in a cell, such as DNA or
RNA, by oxidizing them. On account of its high energy level, ionizing radiation can act on living
tissues from long distances. This feature makes it useful for medical diagnosis. On the other
hand, non-ionizing radiation (such as ultraviolet (UV)) has less energy than ionizing radiation,
therefore does not induce ionization. Moreover, it can only penetrate the surface layers of
animal and higher plant tissues. Effect of non-ionizing radiation depends on the excitation of
electrons in molecules and delivery of its energy to these molecules. If a molecule contains
ionized or excited atoms, these molecules become more reactive than the molecules with
normal stable atoms.

16
UV light does not have enough energy to induce ionization. Organic molecules like purines and
pyrimidines in DNA could absorb UV and become more reactive or reach excited state.
Maximum UV absorption of DNA occurs at 254 nm and the highest level of mutagenity is also
observed at 254 nm.

3. Osmotic Pressure
Osmotic pressure is the force resulting from differences in solute concentrations at opposite
sides on a semipermeable membrane. Osmotic pressure affects bacterial growth, and water
can be drawn into or out of cells depending on the relative osmotic pressure created by
dissolved substances within the cell and its environment. On the other hand, there are
osmotolerant organisms that can grow in solutions with high solute concentration. These
microorganisms have high osmotic pressure tolerance and less requirement for water activity.
Water activity and osmotic pressure are two concepts that affect the salt tolerance. For
instance, halophiles are a group of microorganisms that need a considerable amount of NaCl
in order to grow.

4. pH
Microbial growth is influenced by acidity and alkalinity as well. Optimal pH range for growth
of many microorganisms lies typically in between 6.5-7.5. Yeasts, molds and acid-producing
bacteria create a low pH environment because they elevate the hydrogen ion concentration.
Moreover, pH control is very important for optimal productivity.

Materials:
 E. coli and S. cerevisiae cultures
 Pasteurized milk
 Complex medium
 Medium at various pH
 Medium with various NaCl conc.
 Distilled water
 Bunsen Burner
 Water bath
 Micropipette and tips
 Petri dishes
 Inoculation loop

17
  Glass/disposable cell spreader and test tubes

Method:
Effect of Temperature on Growth
1) Aseptically transfer 1 ml of pasteurized milk into a tube containing 9 ml sterile distilled
water (1:10 dilution) and then transfer 1 ml from 1:10 dilution to another tube
containing 9 ml sterile distilled water (1:100 dilution).

2) From the 1:100 dilution tube (after mixing well), remove 200 μl with a sterile
micropipette and place it on a complex medium containing Petri dish. This is your
control.

3) Divide the contents of 1:100 dilution into two sterile tubes and place one tube at 60oC
and another at 80oC for 5 min.

4) Remove the tubes from water bath. Then pipette 200 μl of each sample onto labeled
Petri dishes.

5) Incubate plates at 37oC for 24 hours. Observe the growth on plates after incubation.

Effect of pH on Growth
1) There are 4 tubes containing nutrient broth at different pH values: 2.5, 5.0, 7.0 and 9.5.

2) Inoculate a loopful of your organism into each tube of pH test broth.

3) Incubate tubes at 37oC for 24-48 hours.

4) Record your results according to the presence or absence of turbidity.

Effect of Osmotic Pressure on Growth

1) There is Petri plates containing agar medium with %5, %10, %15 and without NaCl.

2) Divide plates into two groups so that each group contains an equal number of plates.
Each group will be inoculated with one of the organisms and should be labeled
accordingly. One plate should be a control plate with medium agar only.

3) Use a sterile loop, inoculate E. coli and S. cerevisiae onto agar by streaking.

4) Incubate plates at 30oC for 24-48 hours.

18
Effect of UV on Growth
1) Spread 40 μl of overnight culture on agar plate.

2) A card will be placed on the plate so that it covers the half of

its surface and the plate will be placed under UV light for 10
min.

3) After UV exposure, close the plate and and incubate for 24

hours.

References:

 Atlas, R.M., 1989. Microbiology: Fundementals and Applications, 2nd edition,

MacMillan Publishing Company.
 Alcamo, E.I., 1997. Fundamentals of Microbiology, 5th edition, Benjamin/Cummings
Publishing Company.
 Black, J.G. 2002. Microbiology, Principles and Explorations, 5th edition, John Wiley and
Sons, Inc.
 Johnson, T.R., Case, C.L. 2001. Laboratory Experiments in Microbiology, 6th edition,
Addison Wesley Longman, Inc. California.
 Seeley, H.W., Vandemark, A., Hall, J.S. 1995. Principles of Fermentation Technology,
2nd edition, Reed Educational and Professional Publishing Ltd.
 Snusted, P.D., Simmons, M.J. 2000. Principles of Genetics, 2nd edition, John Wiley &
Sons, Inc.

19
 Experiment 5: Staining Bacteria

Introduction:
Different staining techniques require different types of stains and they can be classified as
shown in Table 5.1.
Table 5.1: Staining techniques and their stain requirements.
Stain Type 1 2 3
Simple Basic dyes Acidic dyes Indifferent dyes
Differential Gram stain Acid-Fast stain -
Structural Endospore stain Capsule stain Flagella stain

In microbiology, synthetic aniline dyes obtained from benzene are generally used. The dyes
are usually salts, although a few are acids or bases, composed of charged ions. The name for
the colored ion is called “Chromophore”. Methylene blue is a typical example as shown below;
Methylene blue chloride  Methylene blue+ + Cl-

Methylene blue+ is the chromophore.

Stains are generally classified depending on the chromophore charges; if the chromophore is
a positive ion, the stain is called as “basic stain” and if negative ion, it is called as “acidic stain”.

Bacteria can be characterized either Gram-positive or Gram-negative as a result of the Gram

staining technique. Staining behavior of bacteria depends on the structure of bacterial cell
wall. In the Gram technique, first, cells are stained with a basic dye. The primary dye is crystal
violet, an aniline dye. The target of this step is the thick-walled bacterial cells. Subsequent
treatment with iodine solution (mordant, Lugol's solution) forms an iodine-crystal violet
complex, which is insoluble in water but soluble in ethanol or acetone, inside the cell.
Therefore, Lugol’s solution causes dye fixing to cell with intensifying the attraction between
the cell and the dye. Next, in the alcohol washing step the multilayer peptidoglycan structure
of the Gram-positive cell wall prevents the iodine-dye complex from being washed out.
Therefore, Gram-positive cells retain the blue stain. On the contrary, iodine-crystal violet
complex is easily removed out of Gram-negative cells owing to the single peptidoglycan layer
present in the cell wall. Finally, a counter dye (such as safranine or fuchsin) having a color
other than blue/violet is applied to cells in order to stain Gram-negative cells and distinguish
them readily under the microscope. This step is called “counter staining”.

Materials:

20
  Overnight Escherichia coli culture
 Overnight Bacillus subtilis culture
 Crystal violet dye
 Lugol’s solution (Iodine agent)
 Distilled water
 Safranine solution
 Decolorization solution
 Bunsen burner
 Microscope
 Inoculation loop
 Culture tubes
 Microscope slides and cover glasses
 Tissue paper

Methods:
Smear preparation on microscopy slides.
1. Flame the inoculation loop until it glows red.

2. Cool it on the walls of your liquid culture tube. In this experiment, you will work on the
cultures of E. coli and B. subtilis.

3. Take a loopfull from each culture and spread the liquid on a small area of clean
microscopy slides per culture.

4. Allow the slides with smear to dry in air.

5. Pass the slides through the hot portion of the flame for 3-4 times.

6. It’s important not to overheat the slides!

21
Simple staining using crystal violet
1. Apply one drop of crystal violet to cover the smears that you have prepared, and allow
it to settle for 3 min.

2. Pour off the stain. Rinse with slowly running water for 30 sec. Do not let the water flow
directly on the smears.

3. Dry the slides by placing them between pieces of tissue paper.

4. Observe your samples under the light microscope (LM) (Use also 100X objective).

Gram staining
1. Cover your smears with Gram’s crystal violet reagent for 1.5 min.
2. Rinse with slowly running water for 30 sec. Do not let the water flow directly on the
smears.

3. Rinse smears with Lugol’s solution (iodine) and pour off the excess. Cover the smears
again with iodine and allow it to remain for 3 min.

4. Rinse with slowly running water for 20 sec. Do not let the water flow directly on the
smears.

5. Apply decolorization solution slowly until dye no longer runs off from the smears (10
sec.)

6. Rinse with slowly running water for 30 sec. Do not let the water flow directly on the
smears.

7. Cover the smears with a drop of safranine solution for 1 min.

8. Rinse with slowly running water for 1 min. Do not let the water flow directly on the
smears.

9. Dry but do not rub.

10. Observe under the light microscope using 100X objective.

References:
 Johnson, T.R., Case, C.L. 2001. Laboratory Experiments in Microbiology, 6th edition,
Addison Wesley Longman, Inc. California.
 Seeley, H.W., Vandemark, A., Hall, J.S. 1995. Principles of Fermentation Technology,
2nd edition, Reed Educational and Professional Publishing Ltd.

22
 Experiment 6: Screening of E. coli and B. subtilis Cells for Antibiotic and
Heavy Metal Resistance by Paper Disc-Agar Diffusion Assay

Introduction:
In 1928 Fleming carried out the first demonstration of selective action of antibiotics in culture
media and discovered that Penicillium notatum inhibited the growh of Staphylococci. Many
years later, Lacey returned to Fleming’s technique and used penicillin to isolate H. pertussis in
1954. Demand for penicillin as a drug increased drastically during World War II resulting in the
purification and commercial production of penicillin in large quantities.

Antibiotics inhibit bacteria via different mechanisms of action. These mechanisms are;
 Alteration of cell membranes
 Inhibition of protein synthesis
 Inhibition of nucleic acid synthesis
 Inhibition of cell wall synthesis
 Anti-metabolic activity or competitive antagonism.

Antibiotics can be divided into bactericidal and bacteriostatic drugs. Bactericidal drugs have
killing effect and cause a decrease in bacterial count. On the other hand, bacteriostatic drugs
merely show an inhibition effect. Bacteria can be naturally resistant to an antibiotic which is
often due to lack of the target molecule in the cell. For instance, mycoplasma is resistant to β-
lactam antibiotics because it lacks the typical peptidoglycan cell wall.
Some metals are crucial for life such as heavy metals working as essential trace elements,
however, most of them are toxic to living organisms at high concentration. Heavy metal ions
that have high atomic masses exert toxicity by forming specific complexes with proteins via
binding strongly to their sulphide groups.

Nickel, cobalt, zinc and copper, which are divalent cations, are important trace elements
whereas, they become toxic, typically through enzyme inactivation, when found in excess.
Heavy metals can also replace other trace elements such as calcium and magnesium bringing
about the instability of biomolecules, genotoxicity and mutagenic effects.

23
Materials:
 Overnight E. coli culture
 Overnight B. subtilis culture
 Incubator
 Bunsen burner
 Micropipettes and tips
 Nutrient broth agar medium
 Petri dishes
 Pre-sterilized Whatman paper discs
 Glass/disposable cell spreader
 Ampicillin
 Kanamycin
 Chloramphenicol
 Heavy metal solutions (Cu, Zn, Co etc.)

Methods:
1. Spread 100 μl of overnight E. coli and B. subtilis cultures on separate agar plates.

2. Soak an individual Whatman paper disc in each antibiotic/heavy metal solution.

3. Place those discs on inoculated agar medium as shown in Figure 6.1.

4. Incubate the plates at 37oC for 16 hours.

5. Measure the diameter of zones of inhibition in order to compare the effects of

different antibiotics and heavy metals on E. coli and B. subtilis cells.

Figure 6.1: Paper disc-agar diffusion assay

24
References:

 Chouldhury, R., Srivastava, S. 2001. Review Article: Zinc resistance mechanisms in

bacteria. Current science. Vol. 81, 768-775.

 Collins, C.H., Lyne, M.P., Grange, J.M. 1989. Microbiological Methods, 6th Edition,
Butterworth &Co Ltd.

 Lombardi, A.T., Garcia, J.O. 1999. An evolution into potential of biological processing
for the removal of metals from sewage sludge. Crit. Rev. Microbiol., Vol. 25, 275-288.

 Olofson, S.K. 2006. Relation Between Drug Exposure and Selection of Antibiotic
Resistant Bacteria, Uppsala Universitet. Retrieved on 5 February 2007.

25