Download Environmental and Food Borne Pathogens Caused by Bacteria Lab

Environmental and Food Borne Pathogens Caused by Bacteria
Lab Part 1A: Culturing and Isolating Bacteria
Introduction:
Environmental and food borne pathogens are particularly Environmental and food borne
illnesses are becoming a greater problem than ever. Every year there is an estimated 47.8 million
food borne illnesses in the US with 128,000 being hospitalized and resulting in over 3,000
deaths. The bacteria we will be looking at are some of the bacteria responsible for these statistics.
They are Escherichia coli, Enterobacter spp., Listeria innocua, Salmonella spp., and
Staphylococcus aureus. See h t t p : / / a g g i e - h o r t i c u l t u r e . t a m u . e d u / e x t e n s i o n / p o i s o n . h t m l for
more information on the pathogenic nature of these and a few other bacteria types.
Many causes of food borne illnesses as well as the illness itself remain poorly understood. It is
estimated that approximately 60% of the outbreaks are caused by unknown sources. This lab will
investigate what bacteria may be involved and introduces the latest technology available for
identifying the causes as well as the treatment. We will discover that due to the latest technology,
treatment for previously deadly outbreaks can be made more readily available in a timely manner
ultimately saving lives.
We will obtain environmental and food borne bacteria from the sources of possible
contamination including soil, surfaces, objects as well as food including meat, poultry, seafood
products, fruits and juices, dairy products, infant formula, chocolate/bakery products, peanut
butter, egg products and animal feed. Students can bring in various sample for us to test with this
lab.
Background:
Bacteria is everywhere. In this lab, we want to concentrate on disease-causing bacteria. To do
this, the bacteria that we are seeking has been limited to four types: Escherichia coli, Listeria
innocua, Salmonella enterica and Staphylococcus aureus. E. coli and Salmonella are examples
of enteric (found in the gut) pathogens that are gram negative. Listeria and Staph. .aureus are
examples of gram positive bacteria. We will use a sterile swab to culture on enriched agar plates
like various possible sources of bacteria. After incubating the plates at 37ºC for 24 hours, we will
isolate the bacterium(a) using a sterile loop, onto another agar plate. We will incubate these
isolated cultures overnight and prepare to identify the bacteria specifically using biochemical
tests.
Purpose:
To culture, isolate and identify a bacteria culture, using the traditional methods similar to those
done in the medical field.
Procedure – First Day:
1.Obtain a sterile swab and an agar plate. Label your plate with your name, period and date on
the agar side (bottom). Swab a possible contaminated sample assigned by the teacher.
2.Innoculate (GENTLY rub the swab on) an agar plate as directed (see diagram) and then using
an inoculating loop to spread over the entire agar plate.
3.Incubate overnight at 37ºC.
Procedure – Second Day:
1. Get agar plate from the incubator. Look for isolated colonies of bacteria. (Ones that are
separated from each other). Ask the teacher for help as this part is very important, especially if
you have more than one colony-type of bacteria.
2. With a new agar plate, use an inoculating loop to touch the bacterial colony and plate for
isolation as follows:
Refer to diagram on left:
Section A: rub initial swab of
sample or loop of bacteria here.
Section B: Flame the loop and cool
and go into A 2 or 3 times and
continue to streak with loop.
Section C and D: Repeat as in
Section B for further isolation.
On ¼ of the plate, gently rub your loop 3 or 4 times across the area
Flame and cool in the agar on the side of the plate. Go into the first section a couple of
times and streak in a narrow zigzag motion
Repeat the previous step two more times with the rest of the sections of the plate.
3.Incubate overnight at 37ºC.
Make a sketch of your plates below: Be sure to indicate if there is more than one type of bacteria
by describing it. Include color, size and if wet/dry.
Agar Plate: First Day
Agar Plate: Second Day
Lab Part 1B: Using Biochemical Testing to Identify Bacteria
Introduction: To identify bacteria, there are some basic tests we can do to identify bacteria. The
bacteria types that are the focus of this lab are divided into two basic types; gram negative and
gram positive. Gram negative bacteria can be subdivided into bacteria found in the gut, called
enteric and those not found in the gut, non-enteric. We will be focusing on two enteric bacteria
that can be distinguished from each other by plating on MacConkey agar. Gram positive bacteria,
such as Staphylococcus aureus and Streptococcus sp. can be can be distinguished from each
other by a presence of catalase test.
Background: See the H/O Part 1B: Understanding Biochemical Testing for Bacterial
Identification
Purpose: For each team’s isolated bacteria, a series of up to five biochemical tests will be done
to definitively identify the bacteria you isolated.
Procedure: The biochemical tests will require 2 days to complete:
Day One:
REMINDER: Label all plates and tubes CLEARLY with your initials, date and period!

Students who have gram negative bacteria will perform Biochemical Test A

Students who have gram positive bacteria, will perform Biochemical Test B

Students who have both bacteria types, perform Biochemical Tests A and B
A. MacConkey Agar: This agar will discriminate between gram negative bacteria (entric
bacteria – found in the gut). If the culture is Escheresia coli from all other gram negative
bacteria. Gram positive bacteria will not grow on this media. E. coli turns bright pink on this agar
whereas all other bacteria will not. Use isolation technique to plate one colony of bacteria on
your plate. If you have more than one gram negative bacteria, divide your plate in half to streak
out the different colonies. Make sure that you use only one colony at a time.
B. Presence of Catalase: Add a few drops of 3% hydrogen peroxide to each culture and look for
foaming that indicates the release of oxygen as a result of hydrogen peroxide breakdown. Add a
few drops of 3% hydrogen peroxide to each culture and look for foaming which indicates the
release of oxygen as a result of hydrogen peroxide breakdown. This test can be used to
differentiate between Staphylococcus aureus and Streptococcus lactis.
Procedure: Day Two and Interpreting Results:
A. MacConkey Agar:
MacConkey agar is a selective and differential medium to isolate enteric bacteria from all other
gram negative bacteria. The agar contains lactose plus a pH indicator that turns red (looks bright
pink) for pH below 6.8. This means the bacteria are able to ferment lactose and the bacteria
colonies will appear bright pink. If the bacteria do not ferment lactose, the colonies remain
normally colored and exhibit no color change. This means they are unable to ferment lactose.
B. Presence of Catalase
Catalase is the name of an enzyme found in most bacteria which initiates the breakdown of
hydrogen peroxide (H2O2) into water and free oxygen. If the bacterium produces the enzyme
catalase, then the hydrogen peroxide added to the culture will be broken down into water and
free oxygen. The oxygen will bubble through the water causing a surface froth to form. This is a
catalase-positive bacterium. A catalase negative bacterium will not produce catalase to break
down the hydrogen peroxide, and no frothing will occur.
Results: see below for how to record your results
For MacConkey agar: (+ = pink, - = colorless)
For catalase: (+ = bubbles, no bubbles = -)
Data Table:
Test
MacConkey Agar
Catalase
Result
Discussion/Analysis Questions:
1. State the chemical nature and function of enzymes.
2. State the function of the enzyme catalase and describe a method of testing for catalase activity.
3. Identify your bacteria based on your biochemical tests as well as colony appearance.
Conclusion Discussion:
Include in your conclusion a discussion of the results of the biochemical test either with the
MacConkey agar or the catalase test. Explain how the test helped you identify your specific
bacteria.
Lab Part 1: Catalase Test is adapted from and used with permission from Dr. Gary Kaiser; Lab 8:Identification of
Bacteria Through Biochemical Testing. See website www.ccbcmd.edu