LABORATORY 3 – THE BACTERIAL GROWTH CURVE

Objectives for Week 3 - After completing these exercises, you should:

1. Be able to follow the growth of a liquid bacterial turbidimetrically.
2. Be able to determine viable cell numbers by plate counts during growth.
3. Understand how to plot and analyze bacterial growth data.

Reading:
Madigan et al (2018): Ch 5: p. 138; 140-143 and 149-152

Pre-lab questions (to be handed in in prior to the start of the lab). The answers must be typed and not
handwritten. [2 Marks]

1. How would you dilute a NaCl solution that has an initial concentration of 15% (w/v) to a 50ml
solution at 0.1%?

2. You have 5ml of an overnight culture (2x108 cells per ml). How would you prepare a series of 1 or
10ml dilutions (maximum dilution 1:100) to obtain a final 200 ml culture at 1x10 3 cell/ml? Show
your work for each step.

3. If 0.1 ml (from a 1 ml sample) of a 105 dilution of pond water was plated and yielded 52 colonies,
how many bacteria were present per ml in the original water sample? Show your work.

4. If 0.1 ml of a urine culture from a 107 dilution yielded 37 colonies, how many bacteria were
present per ml in the original sample? Show your work.

NOTE: Refer to the following site to refresh calculations of dilutions:

http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html

Growth may be defined as an increase in cellular constituents, in some organisms. It leads to a rise in cell
number when microorganisms reproduce by processes like budding or binary fission. In the latter process,
individual cells enlarge and divide to yield two progeny of approximately equal size. Growth also results
when cells simply become longer or larger. However, it is not usually convenient to investigate the growth
and reproduction of individual microorganisms because of their small size. Therefore, when studying
microbial growth, microbiologists normally follow changes in the total population number.

Population growth is studied by analyzing the growth curve of a population in a confined space – broth
culture (for bacteria) or in culture flasks (for tissue culture) for instance. When microorganisms are
cultivated in liquid medium, they usually are grown in a batch culture or closed system. Since fresh medium
is not provided during incubation, nutrient concentrations decline and concentrations of waste products
increase. The resulting curve has four distinct phases (Fig. 3.1).
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Fig. 3.1 Bacterial growth curve in batch culture

Lag Phase: During this phase, cells in a new culture adjust to the medium. Initiation of gene expression and
subsequent increases in enzyme production and cell size occur.

Exponential or Logarithmic Phase: During this phase, metabolic activities proceed at a constant rate, and
cell mass as well as the number of cells double at a constant rate. The time required for the population to
double is called the generation time (g) or doubling time and is usually expressed in hours. Because the
population is doubling every generation, the increase in population is always 2n where n is the number of
generations. The resulting population increase is exponential or logarithmic. The rate of growth during the
exponential phase in a batch culture can be expressed in terms of the growth rate constant (k) or the
number of generations/unit time. k = ln2/g and is often expressed as generations/h.

Stationary Phase: During this phase, waste products that can be toxic or alter the environment (i.e. make
it more acidic) accumulate and/or the availability of nutrients decreases causing the cells to increase their
generation time. Eventually division stops and the population reaches a plateau.

Death Phase: Cells of the population enter this phase when toxic substances accumulate and/or cell
starvation occurs. The rate of decline becomes exponential with time.

Measurement of bacterial population growth can be determined by a number of methods. These include
microscopic counts, plate counts, turbidimetric measurements, nitrogen or dry weight determinations, and
biochemical activity measurements. In this laboratory, you will monitor bacterial growth using
turbidimetric and plate counting methods.

Before coming to the lab, consult the literature to determine the optimum temperature for:
Escherichia coli (DH5)

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You will work in pairs. Since it is impossible to follow an entire growth curve in a 3-hour lab period, most
of your measurements will involve only the exponential and (perhaps) lag phases. To use your laboratory
time efficiently and effectively, there are several things you must do ahead of time.

1. Read the procedures very carefully and be sure that you understand what has to be done.
2. Practice calculating dilutions.
3. Prepare you data tables (i.e. those below) so that you can fill in the data as you track the bacterial
growth.

Table 1: Turbidimetric growth results (Do Not allow the tables to split between pages when preparing
the Lab Report)

Growth Observed Undiluted OD

Tube Dilution Dilution Factor (DF)
time (min) OD600 (calculation)
1 0
2 20
3 40
4 60
5 80
6 100
7 120

Table 2: Plate count results

Growth time Final plated Final plated Final plated Number of Calculated
Tube
(min) Volume (ml) Dilution DF Colonies cfu/ml
1 0 0.1
2 20 0.1
3 40 0.1
4 60 0.1
5 80 0.1
6 100 0.1
7 120 0.1

THE MATERIALS AND PROCEDURES FOR TURBIDITY AND PLATE COUNT MEASUREMENTS (EXERCISES 1
AND 2 RESPECTIVELY) WILL BE DESCRIBED SEPARATELY BUT WHILE YOU ARE PERFOMING THAT LAB, YOU
WILL COLLECT SAMPLES FOR BOTH TYPES OF MEASUREMENTS AT THE SAME TIME. THE PLATE COUNT
STEPS CAN BE CARRIED OUT IN INTERVALS AFTER THE TURBIDIMETRIC SAMPLING AND READINGS.

NOTE: Dilution Factor = Final volume / Solute volume

Example: You want to make a 1:5 dilution of a culture in a final volume of 5 ml  Using this formula, you
would ultimately add 1 ml of your culture to 4 ml of diluent (e.g., LB broth or 0.95% saline solution).

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EXERCISE 1A: Turbidimetric Measurements of Bacterial Growth
In Laboratory 2, you learned how to measure the turbidity of a bacterial culture using a spectrophotometer
(Spec20).

Materials and Methods

Materials:

1. 100 ml of Escherichia coli broth culture (37°C) in LB medium (OD600 = 0.2-0.4).

2. Flask of pre-warmed (37°C) LB broth.
3. 50 ml sterile LB broth to standardize the Spec20 and to dilute the bacterial culture for OD readings.
4. 10 Sterile test tubes (18x150mm) for dilutions.
5. 13mm tubes for Spec20 readings (1 for the blank, others for the samples).
6. Sterile 5 and 10ml pipettes and a P200 pipette with tips.
7. Incubator set at 37°C.
8. Ice in a bucket.
9. Semi-log (1-cycle or 2-cycle) and standard graph paper (not provided).

Procedure

1. Prepare your blank tube (LB medium) and blank the Spectronic 20.
2. Take a sample of 5 ml from your E. coli broth culture provided to you on a flask and make a
measurement (the sample measurement will be your time point zero measurement – record the
reading in Table 1).
3. Remove 100 µl (0.1 ml) from this t0 sample and place it in a microcentrifuge tube (labelled t=0).
Place the tube on ice, it will be used to complete Exercise 2.
4. Place your flask in the assigned incubator (record the time you did this since samples must be taken
at 20 min intervals).
5. In the 20 minutes you are waiting to take the next sample, label the remaining microcentrifuge
tubes for spread plating of the timed samples (you will use these in Exercise 2). You already have
the time 0 sample; you will need 6 more microfuge tubes for the plating exercise (see exercise 2).
Start labelling the microcentrifuge tubes with 20 min intervals, up to the final tube which should be
labeled as 120 minutes (i.e., label the tubes as 20min, 40min, 60min etc.).
6. Place the labeled microcentrifuge tubes on ice.
7. As the 20 min intervals progress, remove two samples (aseptically) each time from the culture flask:
5 ml for the OD600 reading and 100 µl for the spread plate samples in the Exercise 2.
8. Carry out the measuring of the OD600 immediately after removing the 5ml aliquot from the flask: If
the OD600 has not yet reached a value of 0.5, measure the turbidity of the culture directly by placing
the sample directly into a 13mm test tube and placing this tube to the spectrophotometer. Measure
the turbidity and record the OD600 reading and time in the Table 1.
9. Return the flask to the incubator immediately.

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10. If the OD600 of the culture in the flask has reached 0.5 value, you must DILUTE your sample before
taking the turbidity measurement. To do this, dilute your next sample 1:4 (i.e., add 1ml of culture
to 3ml sterile LB broth). Do not return your diluted sample to the main culture flask. You may have
to use a higher dilution for later samples. Be sure to record your dilution factors. NOTE:
measurements of samples with OD600 higher than 0.5 are not accurate; that is why the DILUTION
step is necessary.
11. Stop sampling at 120 min.
12. For each time point: plot OD600 reading (y-axis) vs. elapsed time (x-axis) on semi-log graph paper.

EXERCISE 1B: Plate Count Measurements of Bacterial Growth

Standard Plate Counting Technique (Viable Cell Count)

The standard plate count (SPC) is used to determine the number of living (viable) organisms in various
samples such as water, milk, and foods, and during the various stages of bacterial growth curves. The
technique is simple to perform and produces excellent reproducible results. It is based on the assumption
that each viable cell will form one colony. Thus, the number of resulting colonies is an indication of the
number of viable cells in the original sample. The SPC technique consists of diluting a sample (because the
number of organisms present may be too numerous to count accurately) and then plating the dilutions.
Plating can be performed using either the pour or spread plate techniques; in this laboratory, you will use
the spread plating method. For purposes of accuracy and reliability, after incubation only plates with 30
to 300 colonies are typically counted. (This is a guideline – where there are fewer than 30 colonies, there is
an increased chance of error; where there are >300 colonies, it may be difficult to determine if you are
counting single colonies). The number of organisms in the original sample/ml is determined by multiplying
the number of colonies formed by the dilution factor(s) for the particular plate(s) being used. Plot the
Standard Curve as Log (Viable Cell Count, cfu/mL) versus OD of the sample.

Materials and Methods

Materials:

1. 100 µl samples from Exercise 1.

2. 50mL of sterile saline.
3. 14 LB agar plates.
4. Sterile 5ml and 10ml pipettes.
5. Microcentrifuge or glass tubes
6. Ethanol and a glass spreader.

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Procedure for the preparation of Spread plates (see Fig. 3.2)

1. Label the bottom of all plates with your initials, the appropriate dilutions and the time points.
Pipette the required volume (usually 0.1ml) of the diluted culture onto the medium in the middle
of the plate. Do this in duplicate.
2. Sterilize your glass spreader. To do this:
a. Dip the end of the spreader into ethanol
b. Ignite the alcohol (by passing the spreader through your Bunsen burner flame once)
c. Allow the alcohol to burn away from the flame
d. Let the spreader cool in the air (near the flame, in the sterile region).
3. Check that the spreader is cool by touching the inside of the plate lid.
4. Spread the bacterial sample over the surface of the agar evenly by moving the spreader back and
forth while rotating the plate. Use a turntable or rotate the plate by hand, a quarter turn at a time
on the bench top (your TA will demonstrate).
5. Continue to spread the plate until the surface of the medium is completely dry (this prevents
colonies from “running”).
6. Place the spreader back into the ethanol to avoid contaminating your bench.
7. Invert your plates and incubate them at 37°C for 24 hours.

Fig. 3.2 Spread plating method

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Procedure for counting the viable cell (use the 100µl samples drawn in Experiment 1):

1. Since you can only accurately count plates that have between 30-300 colonies, you will have to
prepare serial dilutions of the 100 µl of samples you collected in exercise 1. Do this as follows:

100µl
sample
collected
in Ex. 1

LB volume 900µl 900µl 900µl 900µl 900µl

Dilution factor 10-1 10-2 10-3 10-4 10-5

100µl 100µl

10-5 10-6

2. Spread 100 µl of the 10-4 and 10-5 dilutions of each sample onto the surface of the LB agar plates.
3. Incubate plates at 37°C for 16-24 hours. Count colony forming units (cfu) the following day.
4. For each time point keep only the plates that have between 30-300cfu. If for a certain time point,
you find that more than one plate contains 30-300cfu, average the counts of these plates and record
this in the table (NOTE: students attending the Thursday Lab, MUST come to observe their plates
on FRIDAY. Please make the necessary arrangements).

Clean-up procedure

1. Clean up the 40x and 100x objective lenses of your microscope using lens paper and ethanol. Repeat
three times using a new piece of lens paper each time.
2. Fill up your distilled water bottle using the distilled water tap located at the sink.
3. Fill up your ethanol squeeze bottle as instructed by your TA.
4. Wipe the surface of your lab bench with ethanol solution.

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ASSIGNMENT: In this assignment, each student must write an independent submission. Follow the
guidelines below to prepare the assignment for Lab 3. The report should include the components listed
below. The discussion section should be brief, and only address what has been requested.

Mark breakdown: [14 marks total] – DUE ONE WEEK AFTER COMPLETION OF LAB 3.

Pre-Lab questions 2 marks

Materials and Methods 0.5 mark
Results:
Tables 1 and 2 (show measurements + calculations) 2 marks
Growth curves 2 marks
Generation time (g) in hours and growth rate constant (k) calculations 1 mark
Standard curves 2 marks
Discussion 4 marks
References 0.5 mark

Detailed Lab 3 Marking Rubric

Materials and Methods [0.5 mark]: Cite lab manual, indicating any changes made.

Tables 1 & 2 [2 marks]: Tables should include your measurements and calculations. They should be neat
and labeled appropriately. Do not include information that is not necessary.

Growth Curves [2 marks]: Drawn by hand on semi-log graph paper. Should be neat, clear, labeled properly.
Plot both OD and viable cell count on one graph. Identify lag phase and/or log phase if possible.

Calculations [1 mark]: Calculate generation time and growth rate constant.

Standard curves [2 marks]: Using linear graph paper, construct the standard curves and produce the graph
by hand and using a computerized method.

Discussion [4 marks; 2 page max]: Brief analysis/explanation of your results. Compare your results to what
has been reported in literature. If relevant, identify possible sources of error. Comment on the accuracy of
your dilutions. Compare your growth curve to that of another bacterium published in peer reviewed
literature* (include a copy of this other growth curve and include a complete reference/citation). Discuss
aspects of these bacteria (environments, physiology) that would lead to differences. *Growth Curve from
the Literature: Include copy of bacterial growth curve figure from research article (published in peer-
reviewed journal), with complete reference citation.

References [0.5 mark]: See p.8-9 for referencing formats.

Pre-Lab questions: 2 marks

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This assignment must be submitted to Turnitin before the deadline in order for it to be marked. An
originality report must accompany your assignment. Failure to submit to TurnItIn and submit the
originality report will result in a 10% per day late penalty.

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LABORATORY 4 – FOOD MICROBIOLOGY AND MICROBIAL GENETICS
Objectives for Week 4 - After completing these exercises, you should:
1. Know how to analyze food for microbial contamination (microbial load).
2. Use differential/selective media to isolate putative enteric bacteria (coliforms) from food.
3. Transform E. coli cells with an R-plasmid by heat-shock.
4. Determine the frequency of E. coli transformation.

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.

EXERCISE
Reading:1: Microbial Genetics (Part 1). Direct DNA Transfer into E. coli Dh5α Cells by Heat
Shock
Madigan et al (2015): Ch 5: pp 144; 149-152 and 155-160.

Introduction: Transformation

During the past decade, the genomes of more than 100 prokaryotic organisms have been sequenced and
annotated. One consequence of all of this genetic information was the discovery that there has been an
enormous amount of shuffling of genes between prokaryotes and between prokaryotic and eukaryotic
organisms. This process is called lateral (or horizontal) gene transfer and it is thought to have played an
important role in both prokaryotic and eukaryotic evolution. Gene transfer between prokaryotic cells can
occur via transduction, conjugation or transformation.

Note that in addition to a chromosome (usually bacteria possess a single, circular dsDNA chromosome),
many bacteria can also possess additional DNA in the form of plasmids, which replicate independently of
the chromosome. (Plasmids are also typically circular dsDNA, but smaller than chromosomes.) Plasmids do
not carry essential genes, but may provide an advantage to the bacterium under certain conditions.

Transformation involves the direct transfer of naked DNA molecules or fragments from one organism to
another. Among the Bacteria, a number of genera of Gram positive and Gram negative organisms are
known to undergo natural transformation, including Bacillus, Streptococcus, Haemophilus and Neisseria. To
take up DNA, the cells must be competent. Competency is a complex physiological state associated with a
particular stage of growth. Competent cells take up DNA and can incorporate a portion of the DNA into
their chromosome via recombination. These cells usually acquire several gene equivalents of foreign DNA,
and are referred to as transformants. One class of transferred genes that are particularly important in
medicine are genes that can give rise to antibiotic resistance. These genes usually encode enzymes that
degrade an antibiotic (e.g., penicillin G and its analogs) or inactivate the antibiotic by modifying its structure
(e.g., streptomycin and chloramphenicol). These genes can reside on either bacterial chromosomes or on
plasmids and are usually associated with mobile genetic elements termed transposons (“jumping genes”).
As the uptake of these genes by transformation can make normally antibiotic-sensitive organisms resistant

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to certain antibiotics, there is a very strong selection for these transformants in environments containing
antibiotics (e.g., hospitals) and they can spread very rapidly.

The Gram negative bacterium (and model organism) E. coli does not appear to be naturally transformable.
However, this organism can be transformed artificially in the laboratory. E. coli cells can be made artificially
competent by treating them in the cold with high concentrations of CaCl2 or RbCl. These cells can then be
made to take up DNA by heat-shocking them at 42oC for 1-2 min. Unlike natural transformation, competent
E. coli cells take up super-coiled plasmid DNA much more efficiently than relaxed circular plasmid DNA or
linear DNA. The transformed plasmid DNA usually replicates autonomously in the cytoplasm rather than
integrating into the chromosome (although some episomes, like the F plasmid, may occasionally integrate
into the chromosome). Electric currents can also be used to temporarily open pores in E. coli cells, and
allow transformation by electroporation. Transformation is one of the most common processes used in
molecular biology to introduce genes of interest into E. coli, Salmonella or other bacteria.

This week, you will perform a transformation experiment using competent E. coli cells and a plasmid (R-
plasmid) carrying a gene encoding resistance to an antibiotic. Antibiotic resistance genes can be very useful
in allowing selection of appropriate bacteria.

Materials and Methods

Materials:

1. One tube of Subcloning EfficiencyTM DH5αTM Competent Cells

2. pBR322 Plasmid DNA (~30µl of ~1ng/µl)
3. 50µl TE Buffer
4. Microcentrifuge tubes
5. 2xLB medium
6. 42°C heat block and a 37°C shaking incubator
7. Pipettors (P200 and P1000), tips and a spreader
8. 10ml Sterile saline
9. 3 LB + Tet plates and 4 LB plates (the LB + Tet plates will be marked with a blue line down the side).

Procedure:

1. Label three microcentrifuge tubes #1, #2 and #3. As you prepare your samples, keep all your tubes
on ice at all times.
2. You will be given one tube containing 60µl of DH5αTM cells that have been thawed on ice.
3. Prepare the following samples:
Tube #1: 25µl of DH5αTM cells and 25µl (25ng) of pBR322 DNA  mix gently by tapping (DO NOT
pipette up and down).
Tube #2: 25µl of DH5αTM cells and 25µl TE Buffer  mix gently by tapping (DO NOT pipette up and
down).
Tube #3: 25µl of TE Buffer only with 25ul pPBR322 DNA
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4. Incubate all tubes on ice for 15 minutes.
5. Heat shock the cells for 45 seconds in 42°C heat block without shaking. This facilitates DNA uptake.
6. Place all tubes on ice for 2 minutes.
7. Add 250µl of 2xLB medium.
8. Incubate tubes at 37°C for 1 hour at 135rpm to permit the cells to express the tetracycline-resistant
gene taken up by transformation.
9. In the meantime, prepare a series of dilution tubes by adding 900µl of LB medium to each of six
tubes and label them 10-1, 10-2, 10-3, 10-4, 10-5, 10-6.
10. Remove the tubes from the shaking water bath.

11. From TUBE # 1:

Add 100µl of the cells to the 10-1 tube you prepared in step 9.
Add 100µl of this dilution to the 10-2 tube and mix.  Continue doing this until you have
completed up to the 10-5 tube.
Use your P200 pipette to create the dilutions and be sure to pipette the bacterial solution up and
down at least 5 times to ensure that the bacteria are resuspended in the medium.
12. Spread 100µl from the 10-1 and 10-2, tubes onto LB+Tet plates  Use a new tip each time.
13. Spread 100ul of the 10-4 and 10-5 tubes onto LB plates without antibiotic
14. From TUBE #2:
Spread 100µl of the undiluted sample on an LB+Tet plate and LB plate (without antibiotic).
15. From TUBE #3:
Spread 100µl of undiluted sample onto an LB plate (without antibiotic).

NB: Prior to plating each dilution (use your P200), be sure to pipette up and down gently to resuspend.
Ensure that the spreader has been allowed to cool down for at least 2 min. Test on the inside of the plate
lid if you are not sure.

Once your transformation plate colonies have grown, count them and fill in the table below (grey indicates
that you will not have a sample for this).

LB+Tet LB
Tube Undiluted 10-1 10-2 Undiluted 10-4 10-5
sample Sample sample sample
#1
#2
#3

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EXERCISE 2: Food Microbiology (Part 1) — Total Microbial Load and Coliform Counts In
Meat Products (This experiment will start while the Exercise 1 is running)
Introduction

Although it is not always obvious, practically all types of foods are contaminated by microorganisms such
as bacteria and fungi. This contamination is often referred to as the microbial load. In some cases, the
presence of microorganisms in food is beneficial while in others it is considered harmful. For example,
certain microorganisms are necessary in the preparation of foods such as cheeses, pickles, sauerkraut,
yogurt and sausage. On the other hand, the presence of organisms such as Escherichia coli, Clostridium
botulinum and Staphylococcus aureus are often responsible for serious and sometimes fatal food poisoning
and toxicity as well as spoilage. Because milk contains carbohydrates, fats, minerals, vitamins and proteins,
and has a pH of approximately 6.8, it is very susceptible to degradation by various species of
microorganisms. Milk, as drawn from healthy cows, contains few microorganisms. However, the milk can
be contaminated by dust and manure in the milking area, the milking machinery and containers, or by the
handlers. Fortunately, these days, the microbial load in most milk is reduced substantially by selective heat
treatments such as pasteurization. Meats are the most perishable of important foods because they contain
abundant quantities of the nutrients required for the growth of bacteria, yeasts and molds. Ground meats
as well as multi-ingredient meat products such as hot-dogs, meat pies and sausage, have higher microbial
loads than whole-meat foods such as steak. In general, the number of organisms in the final meat products
reflects the quality of the ingredients used and the way the products have been handled. In the lab exercise
this week, you will assess the microbial load of a series of meat samples.

Since microorganisms normally associated with animal fecal matter (enteric bacteria or coliforms) should
not be found in food and since some of these organisms are potential pathogens, their presence in food
and water is often monitored as an indicator of microbial contamination. Most coliforms are Gram-
negative, oxidase-negative, non-sporulating, facultative aerobic rod-shaped bacteria that ferment lactose,
and they include members of the genera Escherichia, Salmonella, Shigella, Enterobacter and Klebsiella.
Since coliforms comprise a relatively homogenous group of bacteria, it has been possible to develop growth
media which permit the growth of coliforms while preventing or retarding the growth of some other
organisms (selective medium) and media which distinguish (usually by color) coliform colonies from
colonies of other organisms (differential medium). In this week’s lab exercise, you will use a medium [Eosin-
Methylene Blue agar (EMB agar)] which combines these selective and differential properties to isolate
putative coliforms from meat products. EMB agar contains peptone, lactose, and the dyes Eosin Y and
Methylene blue. Eosin Y and Methylene blue inhibit the growth of Gram-positive organisms and chemically
react under acidic conditions to form a dark purple complex sometimes accompanied by a green metallic
sheen. This metallic sheen is an indicator of the vigorous lactose fermentation typical of certain coliforms.

Important: This week, you will begin the first of several Project Labs where the experiments will continue
for several weeks. For each of these Project Labs, each student will write a single detailed report after the
completion of the project. Since these projects last several weeks and overlap with other laboratories, it

51
is very important that you keep very careful records of your procedures, tests, results, etc. In this first
Project Lab (Labs 4-7), you will determine the total number of microorganisms (microbial load) and the
number of coliforms (Gram-negative bacilli) from a meat (or other food) product. You will also characterize
and eventually identify the genus (and perhaps species) of an unknown coliform which you will be given.

Materials and Methods

Materials

1. Meat sample (i.e. SPAM – canned meat, ground beef stored at 4°C for 3 days, ground beef stored
at 37°C for 3 days)
2. 4 LB agar deeps (20ml; melted and stored at 50°C)
3. 3 EMB agar deeps (20ml; melted and stored at 50°C)
4. 1 EMB agar plate
5. One 99 ml sterile saline dilution bottle and one 9.9 ml sterile saline bottle
6. Sterile weighing paper
7. Forceps, spatula, P200 and P1000 pipettors
8. 7 sterile Petrie plates and sterile 18mm test tubes
9. Ethanol container
10. Gram stain kit, microscope slides and cover slips
11. Controls for Gram stain (E. coli and B. subtillis)

Procedure

1. Aseptically weigh out approximately 1g of your meat sample. To do this:

a. Sterilize your forceps by dipping the tips into ethanol and igniting the alcohol by passing the
forceps through the flame once. Wait until the ethanol burns off and let the forceps cool in the air.
b. Pick up on piece of sterile weighing paper with your forceps and place it on the tray of the
balance. Zero the balance.
c. Sterilize the end of your spatula in ethanol, allow it to cool, and transfer about 1g of meat to the
weighing paper. Record the exact weight in your lab notebook.
2. Touching only the corners of the weighing paper with your fingers, transfer the meat to the sterile
99ml saline dilution bottle. Push the meat off the paper with a sterile spatula and cap the bottle.
3. Mix the meat thoroughly in the water by shaking the bottle vigorously about 5 min. Allow the
particles in the sample to settle. This is a 10-2 dilution of your sample.
4. Prepare a 10-4 dilution of your sample by transferring 0.1ml (1:100) of your 10-2 dilution to the 9.9ml
sterile saline in a tube. Use a P200 pipettor. Mix the contents of the tube well by vortexing.

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Plating Cells for Total Counts (Microbial Load) using pour plate technique

1. Label the bottoms of four sterile Petri plates with the dilutions 10-2, 10-3, 10-4 and 10-5 (include your
initials and lab section on each plate).
2. Make the following transfers as accurately as possible:
1.0ml of the 10-2 dilution to the 10-2 plate
0.1ml of the 10-2 dilution to the 10-3 plate
1.0ml of the 10-4 dilution to the 10-4 plate
0.1ml of the 10-4 dilution to the 10-5 plate
3. One plate at a time, pour the nutrient agar deep into the plate and immediately rotate the plate to
mix the diluted sample with the melted agar. Your TA will demonstrate how to mix your samples.
Place each plate with the lid facing up on your bench top and let the agar solidify.
4. Incubate your plates inverted at 37°C for 24 hours, or as directed.

Plating Cells for Coliform Counts

1. Using the streak plate method from lab 1, streak a large loopful of the sample from the 10-2 dilution
bottle on the EMB agar plate. Label the bottom of the tube with your initials and lab section.
2. Label the bottoms of three sterile Petri plates with the dilutions 10-2, 10-3, and 10-4 and your initials
and lab section.
3. Make the following transfers. You will use pour plating technique.
1.0ml of the 10-2 dilution to the 10-2 plate
0.1ml of the 10-2 dilution to the 10-3 plate
1.0ml of the 10-4 dilution to the 10-4 plate
4. One plate at a time, pour the EMB agar deep into a plate and immediately rotate the plate to mix
the contents. Let the medium solidify in the plates.
5. Incubate the plates inverted at 37°C for 24 hours, or as directed.
6. Discard your plates after you have made your observations.

Gram Stain

1. Perform the Gram stain experiment for the control organisms given to you.
2. Perform a Gram stain using the 10-2 dilution of your meat sample, and describe the results (compare
with the above control experiment). All group members should look at the microscope cover slip
and record the observations.

Before coming to your next lab, you must

A. Count the colonies on your nutrient agar and EMB plates. Coliforms are potential pathogens; DO
NOT OPEN THE PLATES!). When finished counting, discard the plates in the appropriate bags.
B. Use the appropriate factors to determine the total count (microbial load) and coliform count in your
original meat sample.

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C. Express your results as the number of microorganisms/ gram of meat. Remember to only use plates
containing between 30-300 colonies (>300 can only be used if the colonies are distinct).

NOTE: The results obtained from Exercise 1 will be used to prepare the assignment in Laboratory 5. (p. 61).
You must obtain your classmate’s data for this report as well.

Clean-up procedure

1. Clean up the 40x and 100x objective lenses of your microscope using lens paper and ethanol. Repeat
three times using a new piece of lens paper each time.
2. Fill up your distilled water bottle using the distilled water tap located at the sink.
3. Fil up your ethanol squeeze bottle with the ethanol as instructed by your TA.
4. Wipe the surface of your lab bench with ethanol solution.

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ASSIGNMENT (Microbial Genetics): In Lab 4 you will prepare an assignment only for Exercise 1, DNA
Transformation. The assignment will be worth 14 marks (it will be converted to a mark out of 7 at the end).
IT IS DUE TWO WEEKS AFTER YOUR LAB. The questions that need to be included in this assignment are
provided below. The assignment should not exceed 4 pages.

1. Use the information on transformation efficiency discussed in lecture, your text book and in any
other primary sources (being sure to reference everything!) to answer the following questions:
a. What does competent mean with respect to cells about to be transformed? [1 mark]
b. How does natural competence differ from artificial competence? What might you expect if
chemicompetent cells were used for electroporation? [2 marks]
c. Would you consider E. coli to be naturally competent? Explain why or why not by looking at
the process at a molecular level. [1.5 marks]

2. Prepare a data table with the number of transformants similar to the one set up in this manual
(when creating the table do not forget to consider the final dilution on each plate!). Would you
consider your experiment a success? Support your answer with direct references to your data
(include an explanation of the controls you used and their purpose). [4 marks]

3. The average molecular weight of a nucleotide (base + deoxyribose + 1 phosphate group) in DNA is
308g/mol of 308 Daltons (Da). This number is the average molecular weight of
A = 312.2 g/mol
G = 328.2 g/mol
C = 288.2 g/mol
T = 303.2 g/mol

a. Which plasmid was used for the transformation experiment? What is the size of this plasmid
(in kilo base pairs; kbp)? How many bases is this? [1 mark]
b. What is the molecular weight of one plasmid molecule? [0.5 mark]
c. You used 25µl of 1ng/µl plasmid DNA per transformation. How many grams of plasmid DNA
did you use? How many moles of plasmid DNA is that? How many molecules of plasmid DNA
is that? [1 mark]
d. Assuming each transformed cell pick up one plasmid molecules during the transformation
process, what percentage of DNA molecules successfully entered and replicated inside the
competent cells? [1 mark]
4. What results would you expect if Tetracycline was omitted from the plates? Why? Would the
results of your experiment change if Ampicillin was used instead of Tetracycline? Why? [2 marks]

This assignment must be submitted to Turnitin before the deadline in order for it to be marked. An
originality report must accompany your assignment. Failure to submit to TurnItIn and submit the
originality report will result in a 10% per day late penalty.

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Plasmid map

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