LABORATORY 5 – Food Microbiology and Microbial Genetics

Objectives for Week 5 - After completing these exercises, you should:

1. Know how to analyze isolated organisms for catalase and oxidase activity.
2. Know how to prepare a lysate of the Escherichia coli transducing phage, P1.
3. Know how to run PCR gel.

Reading:
Madigan et al (2018): Ch 4: p.108-109; Ch 11: p. 316-320; Ch 28: p.835 and 849; Ch 32: p.943-955.

NB: This week (and in the remaining weeks), you will be working on more than one experiment/project at
the same time. Therefore, it is essential that you:

1. Read over the laboratory procedures carefully before coming to the lab.
Reading:
2. Comeet
Madigan prepared toCh
al (2015): start work
5: pp immediately.
144; 149-152 and 155-160.
3. Organize your time efficiently.
4. Keep detailed notes on your results and observations for each experiment/project.

EXERCISE 1: Microbial Genetics (Part 2). Phage Transduction of E. coli.

Introduction: Generalized Transduction

As discussed in the previous lab, gene transfer between prokaryotic cells can occur via transduction,
conjugation or transformation. In the process of transduction, gene transfer is mediated by a
bacteriophage (or phage), a virus that infects a bacterial host.

Bacteriophages have different types of cycles:

Lytic/Virulent – Virulent viruses kill hosts after infection. These are the most commonly known
bacteriophages. (NB: “virulent” is used differently when we talk about bacteriophages than when we
are talking about microbial pathogens). Examples: T-phages.

Lysogenic/Temperate - Some bacteriophages may not immediately generate new progeny through lytic
cycle. In lysogeny, most viral genes are not expressed, and the genome or the virus is replicated along
with host genome. The prophage (provirus) is the latent form of the temperate virus (usually the
genome). Bacteria containing prophages are called lysogens. Lysogens retain potential for virus
production – under appropriate conditions, induction of the lytic cycle can occur. Examples: Lambda,
P1, P22.

Chronic/Continuous: A chronically infecting phage (or virus) can release progeny into the environment
without killing the host cell. Example: M13

If the phage transfers specific genes at high frequency the process is called specialized transduction,
whereas the random transfer of genes at a low frequency is generalized transduction.

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Transduction Types

Generalized Transduction: Bacterial cells are killed in lytic infections, and on occasion, bacterial DNA is
mistakenly packaged into a new phage head (rather than phage DNA). The resulting transducing particle
may be able to inject the DNA into another bacterium. In some cases, the transferred DNA may be
incorporated into the chromosome by homologous recombination, replacing the existing genes and
possibly changing the genotype of the recipient. More than one gene may be transferred (cotransduced),
depending on how much DNA the phage head can hold.

Specialized Transduction: This type of transduction requires lysogeny. Bacteriophages that integrate into
the bacterial chromosome may inaccurately excise. When this occurs, some of the prophage is left behind,
and some host bacterial genes are taken. All resulting phage particles will contain a mixture of phage and
bacterial DNA. These particles may transfer the DNA to a new host cell, and if recombination occurs, the
transferred bacterial genes may be exchanged.

Many transducing particles are defective (non-infectious) and require replication-competent helper phage
to replicate, depending on which phage genes are present/lost.

Over the next four weeks, you will use Escherichia coli and the bacteriophage P1 to demonstrate lateral
gene transfer by generalized transduction. This week, you will grow P1vir phage on the donor strain by
making a phage lysate (a lysate should contain only bacteriophage, not bacterial cells). Next week (Lab 6)
you will titer your phage lysate, and in the third (Lab 7) and fourth weeks (Lab 8), you will perform the
transduction and analyze the transductants for the transferred genetic material. (NB: P1 is a lysogenic
phage. To ensure that the transformed cells do not become lysogenic, mutants of P1 that cannot lysogenize
such as P1vir or P1kc are often used in generalized transduction experiments.)

Materials and Methods

Materials:

1. Overnight culture of E. coli (JW0234-1) in Luria Bertani (LB) broth supplemented with 5 mM CaCl2
2. Two 6ml tubes of R-top agar, melted and kept at 50°C
3. Two R-agar plate kept at 37°C
4. Lysate of P1vir; Use 100 µl of 1:50 dilution, and 100 µl of 1:100 dilution
5. 13mm glass tubes

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Procedure to grow P1vir phage on the donor strain:

1. You will receive an aliquot of E. coli JW0234-1 overnight culture.

2. In a 13mm glass tube, preadsorb the P1vir phage to the cells by adding 0.1ml phage to 0.1ml of the
overnight culture. Incubate the tube a 37°C incubator without shaking for 30min.
NB: R-top agar should be kept in boiling water bath up until about 5 min before adding to the
bacteria/phage mix. You must be able to hold the agar tube in your hand before adding it to your
sample to avoid killing the bacteria. If you take R-top out too early it might start solidifying. In this
case, put the tube back into water bath to allow the agar to melt again. Be careful that it is not too
hot before adding to the bacteria/phage mix.
3. Once cooled to the appropriate temperature, add 6ml of R-top agar to the bacteria/phage mix (not
vice versa) and mix gently (no bubbles).
4. Immediately pour the contents onto an R plate (not an empty plate). Swirl the plate gently to ensure
that the R-top agar is evenly distributed over the whole surface of the R-agar. You must work fast
as the R-top agar cools quickly.
5. Place the plate on your bench top (lid up) to give the agar the chance to solidify.
6. Incubate the plates overnight 37C. You must come back the next day to observe your plate. After
the overnight incubation, transfer your plate to 4°C
THE FOLLOWING WEEK (ONE DAY BEFORE YOUR NEXT LAB) Your TA will add 6ml of LB+Ca2+ to
your plate and place it back at 4°C.

NOTE: No assignment for this Exercise. The experiment continues in Lab 6 and 7.

EXERCISE 3: Food Microbiology (Part 2). Characterization of the Unknown Coliform (Work
in pairs)
Food products contain a wide variety of microorganisms, many of which are capable of growing on EMB
agar. To determine whether a particular EMB positive microorganism is a coliform and to classify it with
respect to its genus and species, isolated colonies of the organism are usually Gram stained and subjected
to a battery of biochemical tests.

Two simple biochemical tests commonly used to classify microorganisms are the catalase and oxidase
tests. These tests determine if a particular organism can tolerate molecular oxygen (O2) and use
cytochrome c to transfer electrons to O2 during aerobic respiration. Certain forms of molecular oxygen such
as the superoxide radical (O2•) are toxic to all living cells. Aerobic organisms can detoxify these dangerous
forms of oxygen (most possess a superoxide dismutase enzyme that adds protons to the superoxide radical
to form hydrogen peroxide):

2O2 + 2e- 2O2• + 4H+ 2H2O2

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Since hydrogen peroxide itself is also toxic, most aerobic microorganisms produce the enzyme catalase,
converting this chemical into water and oxygen.

2H2O2 2H2O + O2

Most enteric bacteria are catalase positive.

Some (but not all) aerobic microorganisms use the electron transport chain (ETC) to transfer e- to O2 during
respiration. To determine if this pathway is being utilized, microorganisms can be tested for the presence
of the enzyme oxidase. In this test, if oxidase is present, electrons are transferred from the reduced form
of the substrate tetramethyl-p-phenylenediamine (TMPD) to the oxidized form of cytochrome c, which will
be available to accept electrons if the ETC is functioning. The oxidized form of TMPD forms a dark violet or
purple color (oxidase positive) while the reduced substrate remains colorless (oxidase negative). The
oxidase test is used in clinical laboratories to distinguish between the oxidase positive organisms such as
Pseudomonas, Neisseria and Vibrio and the oxidase negative “enterics”. This week, you will carry out Gram
stains, catalase tests and oxidase tests on an unknown to determine if it is a possible coliform.

Materials and Methods

Plate counts

Before beginning to analyze the putative coliform, each group should record their total counts (from
Nutrient agar) and total coliform counts (from EMB agar from week 4) in the table on the blackboard.
Results should be expressed as the number of microorganisms/g of meat. Be sure to indicate, how your
meat was treated. Discuss the results obtained by all of the groups in the Discussion section of your Project
report.

Materials:

1. Gram stain kit (for the control Gram stain experiment)

2. Control bacteria for Gram staining
3. 3% solution of H2O2
4. 1% solution of tetramethyl-p-phenylenediamine (TMPD) with a dropper
5. Microscope slides
6. Wax pencil
7. Liquid culture of E. coli (catalase positive control)
8. Nutrient agar streak plate of Pseudomonas fluorescens (oxidase positive control)
9. Liquid culture isolated from the meat sample

Procedure:

1. Conduct a Gram stain on the positive and negative control samples and the unknown sample (the
liquid culture from the meat).
2. Conduct the Catalase and Oxidase tests as follows:

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 Catalase reaction: Remove 0.5ml from the 5ml liquid culture provided and place in a clean 18mm
test tube. Add 0.5 ml H2O2 and vortex for 20-30 seconds to mix well.

Oxidase reaction: Remove 1.125 ml of the 5ml liquid culture provided and place in a clean 18mm
test tube. Add 50 µl of 1% α-naphtol and 75 µl of 1% TMPD. Vortex for 20-30 seconds to mix
well.

Assignment (Food Microbiology Parts 1 and 2): In this assignment you will include the results from Exercise
3 in Lab 4 and Exercise 3 from this lab (Lab 5). Do not exceed 5 pages total. The assignment is worth 14
marks [it will be converted at the end to a mark out of 7]. IT IS DUE ONE WEEK AFTER YOUR LAB 5.

1. Title page: Should include a descriptive title (simply writing Meat Assignment is not a proper title)

2. Materials and Methods: Refer to the lab manual and only note changes. [0.5 mark]

3. Results: [7 marks]
a. In table format, describe the Gram stain and streak plate observations of your meat sample.
Provide a hand drawn image of what you see. [1 marks]
b. In table format, provide your colony counting results for the nutrient agar and EMB plates. Be
sure to include the dilution factor and colony count. [1 mark]
c. In table format, provide the class results for all three meat samples. Include microbial load and
coliform count. Include a sample calculation. [2 marks]
d. In table format, describe the catalase and TMPD test results. [1 Mark]
e. Provide a written description of all results. [2 marks]

4. Discussion: [6 marks]: Incorporate answers to the following points (discussion should not be
presented as a series of bullet points!)
 Do the observed results correlate with what was expected? Provide examples from
primary literature to support your findings.
 What type of treatment(s) was the SPAM meat sample likely subjected to? Discuss how
these treatments along with the two others influenced microbial load of the meat
products.
 Besides the catalase and oxidase tests you conducted as part of this lab, what other
biochemical/ biophysical experiments to microbiologists use to identify microbial
pathogens? (Include references).

5. References: All sources must be properly references in text and at the end of your paper. [0.5
mark]

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 LABORATORY 6 – MICROBIAL GENETICS (PART 3)

Objectives for Week 6 - After completing these exercises, you should:

1. Titer a P1vir lysate.

Reading:
Madigan et al (2018): Ch 4: p. 106-109; Ch 11: p. 316-320; Ch 28: p. 835

EXERCISE 1: Assessing the viral titer following transduction

Generalized Transduction (continued)

Last week you made a P1vir phage lysate by growing the phage on your E. coli donor strain. When using
P1vir (or P1kc) in transduction experiments it is necessary to prevent excessive killing of the recipient strain.
This can be done by using a very low multiplicity of infection (m.o.i.; the ratio of phage particles:bacterial
cells) such as 1:100. This ensures that a cell infected by a transducing particle (P1 carrying bacterial DNA)
is unlikely to be superinfected and lysed by a normal P1 phage particle. To do this, it is essential to know
the approximate concentration of phage in your lysate. Therefore, this week you will titer your P1vir lysate.
You will determine the titer that will result in an adequate number of plaques (~50-300 plaques). Do not
use plates that have no E. coli growth as this suggests that your titer is too high.

Materials and Methods

Materials:

1. P1vir lysate
2. A fresh overnight culture of an E. coli strain for titering (MG1655).
3. 10ml of sterile phage dilution buffer.
4. Six 13mm tubes containing 6ml of R-top agar (melted and stored at 95°C).
5. Six R-agar plates.
6. Sterile tubes, 6 microcentrifuge tubes for dilutions and 6 small tubes for incubations.
7. Sterile pipettes
8. Chloroform
9. Two 50ml centrifuge tubes
10. Cell scraper

Procedure:

1. Describe the appearance of your lysate plate and record it in your lab notebook.
2. Choose the plate (either 1:50 or 1:100) with the most number of plaques for this experiment and
discard the other one.
3. Using a cell scraper (flat blade cell lifter), loosen the entire upper layer of soft agar and transfer
the agar along with all of the LB + Ca2+ broth to a 50 ml centrifuge tube.  Remember the TA

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 added this broth to your plate the day before this lab. If all this buffer was absorbed, add an
additional 2 ml of LB + Ca2+ to ensure proper phage recovery.
NB: When scraping, do not scrape too hard as this will detach the R-agar as well. You only want to
remove the R-agar overlay.
4. Add 5 drops of chloroform and vortex the tube for 30 seconds. Centrifuge the tube a 4,150rpm for
30 minutes. BE SURE TO BALANCE YOUR TUBES.
5. Carefully pipette the supernatant into a microcentrifuge tube (~1ml). DO NOT transfer any of the
chloroform (chloroform will be the bottom layer).
6. Spin for 2 minutes at 13,300rpm.
7. Using your P200 pipettor, remove the upper 1ml and transfer to a clean microcentrifuge tube. If
needed, leave some supernatant behind to avoid disrupting the pellet.

8. YOU NOW HAVE YOUR P1VIR LYSATE. Label it with your name, your lab partner’s name, lab section,
and TA name and give it to your TA (at the end of the lab).

9. Prepare serial dilutions of your phage lysate using phage dilution buffer and sterile tubes. Make
dilutions of 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6. The final volume for each dilution should be 1ml. You
must show your calculations for creating these dilutions in your lab notebook and lab report.
10. Preadsorb 0.1ml of each phage dilution with 0.1ml of E. coli in 6 small glass tubes at 37°C for 30
minutes.
11. After 10 minutes, add 6ml of melted R-top agar to each tube one at a time (be sure to let it cool),
mix gently, and immediately pour the entire content of the tube onto an R-agar plate. Immediately
rotate the plate to spread the R-top agar evenly. Place the plate on the bench with the lid up to let
the R-top agar solidify.
12. Incubate the plates at 37°C (lid side down) overnight. Do not incubate longer than 2 days,
13. Examine the plates BEFORE your next lab session and count the E. coli colonies on the plates. Use
the plates with less than 300 colonies if possible.
14. Once you and your lab partner have observed the plates, throw them out.

Note: No Assignment is due. The experiment continues in Lab 7 and the result will be used to prepare the
assignment in Lab 7.

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 LABORATORY 7 – MICROBIAL GENETICS (PART 4)

Objectives for Week 7 - After completing these exercises, you should:

1. Be able to perform a generalized transduction of Escherichia coli using phage P1vir.

Reading:
Madigan et al (2018): Ch 4: p. 106-109; Ch 11: p. 316-320; Ch 28: p. 835

EXERCISE 1: Phage P1vir transduction of E. coli

Reading:
Introduction: Generalized Transduction (continued)
Madigan et al (2015): Ch 5: pp 144; 149-152 and 155-160.
During generalized transduction, bacterial genes are transferred randomly from donor to recipient cells at
a low frequency, e.g., 10-5. Not only can generalized transduction be used to demonstrate lateral gene
transfer, it can also be used to determine the approximate distance between genes (fine-structure
mapping). In fact, the E. coli chromosome was originally mapped using this technique.

In the following exercise, you will use P1vir transduction to demonstrate the lateral transfer of the proA
gene from the donor strain JW0234-1 to the recipient strain NK5525 (both are E. coli strains). The genotypes
of the two strains are provided below. Strain JW0234-1 contains the proA gene (5.62 min away from the
origin of transcription in the chromosome, that encodes for the gamma-glutamylphosphate reductase
enzyme, which is involved in Proline biosynthesis. Next to this gene, at 5.66 min away from the origin of
transcription in the chromosome, a kanamycin resistance gene (kan) is present. After JW0234-1 is infected
by the p1vir phage, the phage progeny will randomly contain multiple parts of the JW0234-1 chromosomal
DNA some of which will contain the proA gene (and the kan gene). Upon p1vir infection of the NK5525
recipient cells, [which are tetracycline resistant (tetR)], the phage should insert the proA and kan genes in
the NK5525 chromosomal DNA. Successful incorporation of the proA and kan genes into the NK5525
chromosomal DNA will lead to inactivation of the tetracycline resistance gene. This in turn means that the
cells are now tetracycline sensitive (tetS)

Donor: E. coli JW0234-1  proA+, kanR

Recipient: E.coli NK5525  proA-, tetR

R = Resistant

Materials and Methods

Materials:

1. Pellet of NK5525 resuspended in 5ml LB buffer containing 100mM MgSO4 and 5mM CaCl2
2. P1vir lysate of JW0234-1 (you prepared the lysate by infecting this JW0234-1 E. coli strain in lab 6)
3. 2 small sterile tubes
4. 1ml of 1M sodium citrate
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 5. 4 LB agar plates supplemented with tetracycline
6. 4 LB agar plates supplemented with kanamycin
7. 10ml Saline solution
8. 10ml Phage dilution buffer

Procedure:

1. Calculate the titer of your original lysate and express it as phage/ml.

Plaque forming units (PFU) = Number of plaques

Dilution x Volume of diluted virus added to plate

** You need to do this to determine how much of the viral lysate you will require **

2. Briefly vortex the tube containing your NK5525 suspension to ensure a uniform mixture.
3. Label 4 small tubes as follows:
Tube 1: undiluted phage + cells (NK5525)
Tube 2: Diluted phage (1:50 dilution in phage dilution buffer) + cells (NK5525)
Tube 3: Diluted phage (1:100 dilution in phage dilution buffer) + cells (NK5525)
Tube 4: cells only (NK5525) + saline buffer

Tube Phage E. coli cells

#1 100µl 100µl
#2 100µl 100µl
#3 100µl 100µl
#4 0µl 100µl + 100µl of saline

NB: If you determine your titer to be high, you might consider increasing the dilution above 1:50 or
1:100 (e.g. 1:500 or 1:1000)

4. Add 100µl of P1vir phage to and 100µl of NK5525 cells to tube # 1, 2 and 3.
5. For tube #4: add 100µl of NK5525 cells and 100µl of saline buffer.
6. Incubate both of the tubes at 37°C (without shaking) for 25 minutes to preadsorb the phage.
7. Add 200µL of 1M sodium citrate to all four tubes to prevent readsorption of phages/ excessive
killing.
8. Plate the entire contents of tube #1, 2 and 3 onto a LB agar supplemented with kanamycin.
9. Using tube #4, prepare four serial dilutions (10-5, 10-6, 10-7 and 10-8) in saline solution (1ml final
volume in microcentrifuge tubes).
10. Plate the entire contents of each of these tube #4 serial dilution samples onto a LB agar plate
supplemented with Tet.
11. Also plate the remaining tube #4 content onto a LB agar plate supplemented with kanamycin.
12. Make sure the inoculated material on the plate has dried before incubating.
13. Incubate plates at 37°C with the lid down for 24 hours. If colonies are not visible, incubate for an
additional 24 hours.
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 Tube Kanamycin Tetracycline
#1 Yes No
#2 Yes No
#3 Yes No
#4 Yes (NK5525 cells + saline) Yes (all 4 dilutions)
To determine your % of transduced cells use the following formula:

Transduction frequency = # of transductants (# of colonies on the kan+ plate) x 100

+
Total number of colonies grown x DF (based on the tet plates)

ASSIGNMENT: The format of this assignment and the mark breakdown are provided below.

Mark breakdown: [10 marks] – ASSIGNMENT IS DUE ONE WEEK FOLLOWING COMPLETION OF LAB 7

1. Introduction [2 marks]
a. Include a short description of the wild-type P1 viral cycle, and how P1vir differs from the
WT phage.
b. Describe the role of the proA gene in E. coli (i.e., what does the resulting protein do?).
2. Materials and Methods [1 mark]
a. Refer to the lab manual; include modifications (if any).
3. Results [3 Marks]
a. Indicate the titer of your lysate and calculate the transduction frequency you obtained.
b. Show all calculations and dilutions you had to make to perform the generalized
transduction.
4. Discussion [4 Marks]
a. Briefly discuss your results in terms of what would have been expected.
b. Suggest another technique that is used for studying genes in bacteria (with appropriate
citation of primary literature source).

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