WEEK 7-8: BACTERIAL Techniques in Lab

ISOLATION ● Colony screening

● Sterile technique
Synopsis ● Plate streaking
In the Bacterial Isolation simulation, you will
investigate the cause of a contamination of Theory
poultry meat by a dangerous bacteria strain Salmonella
that is resistant to common antibiotics. After Salmonella is a genus of rod-shaped, gram-
taking samples from the chicken farm, you will negative bacterium. Salmonella is usually found
work in the virtual laboratory to isolate single in the digestive tract of animals. Salmonella is
colonies of the deadly bacteria among a variety one of the most common causes of food
of different species. To do so, you will learn poisoning, it causes a condition called
how to work under sterile conditions, and you salmonellosis.
will be able to practice and perfect your plate
streaking technique.

Identifying ampicillin resistant bacteria

The premise of the Bacterial Isolation lab is a
report of ampicillin-resistant bacteria in poultry
meat. You will visit the place of origination, a
chicken farm, in hopes of identifying the
bacteria strain. The first task is to take a sample,
which will contain a variety of bacterial strains, The genus Salmonella belongs to the same
and from that, you must identify which strains family as the genus Escherichia. Both are
are resistant to ampicillin by isolating single enteric bacteria adapted to grow in the gut of
colonies. their host. Therefore, their optimal growth
temperature is around 37° C. At 4° C bacterial
Aseptic technique growth is halted, but the bacteria are not
In the Bacterial Isolation lab, you will learn how killed off.
to use aseptic techniques—for example,
remembering to turn on the Bunsen burner and Accordingly, this is why it is important to store
sterile their loop in between streaks. food in the refrigerator—to prevent any
potential Salmonella bacteria from
Plate streaking technique accumulating. Additionally, before
In order to identify the specific bacteria strain, consumption, food should be heated to 75° C
you will have to perform bacterial isolation for at least 10 minutes to kill o any traces of
using the plate streaking technique. There is an Salmonella that may be present.
unlimited supply of agar plates, giving you the
opportunity to practice this technique as many When working with Salmonella in the lab,
times as you like. Results are given immediately, Biosafety level 2 (BSL 2) should be followed. All
as opposed to waiting a full 24 hours for of the instruments that come into contact with
incubation, as when performed in reality. You the bacteria must be sterilized and all work with
will also streak a special Salmonella Shigella the bacteria should be conducted in a
agar. The Salmonella Shigella agar contains a designated workspace.
certain medium that only promotes growth of
Gram-negative strains. Each of the different Salmonellosis
strains will exhibit a certain phenotype when Salmonellosis is an infection caused by
grown on the Salmonella Shigella agar. Salmonella bacteria.

Learning Objectives Salmonella is one of the most common causes

At the end of this simulation, you will be able of food poisoning. Typically, ingested
to... Salmonella cells are killed by the acidity of the
● Understand the importance of bacterial stomach; however, Salmonella has evolved to
growth for the investigation of pathological possess a degree of resistance to stomach
microorganisms acidity. As a result, surviving Salmonella cells
● Work under aseptic techniques multiply in the small intestine and release
● Understand the concept of a single colony endotoxins upon cellular death. These
● Perform plate-streaking techniques endotoxins poison nearby cells and trigger
● Use selective media for isolation purposes inflammation, which leads to diarrhea,
vomiting, cramping, and fever.
Salmonella infections usually last for 4-7 days,
and most patients recover without needing any
treatment. In some cases, the diarrhea is so
severe, that the patient needs to be
hospitalized, and provided with fluids. Other
severe complications occur when Salmonella
spreads to the bloodstream and infects other
organs, a process which is termed sepsis. If such
a patient is not treated with antibiotics
immediately, this systemic infection can be
fatal. Infants, seniors and people with impaired
immune systems are more likely to have a
severe reaction to Salmonella.
Salmonella outbreaks
It is estimated that 93.8 million cases of
Salmonellosis occur every year. Most of them
are caused by food-borne ingestion of
Salmonella bacteria. In the 1980's, a significant
increase of Salmonella cases was recorded in
chicken meat, eggs, and egg products. This
emerging public health problem was likely the
result of declining genetic diversity in chickens
farmed with modern poultry practices.
Salmonella Shigella agar
Culturing techniques are an efficient way to
identify bacterial strains depending on the
bacterium’s growth requirements. The growth
medium’s composition can be designed to
restrict the growth of certain strains while
simultaneously allowing other specific strains to
grow.
The Salmonella Shigella agar (SSA) contains
peptone, lactose, and all other essential micro-
nutrients. Bile salts and brilliant green dye act
as selective agents as they allow for only the
growth of certain gram-negative strains and
prevent the growth of gram-positive strains.
The medium contains the pH indicator neutral
red, this indicator is orange under neutral
conditions but changes to red when the pH
drops below 6.8. The lactose fermenting
bacteria, Escherichia coli, can cause this change
when lactose is converted into lactic acid, as the
pH is decreased and the pH-indicating
molecules in close proximity to the cells turn
red. Salmonella, however, cannot ferment sugar
and uses peptone as a carbon source.
As an additional mechanism for identification,
the medium contains ferric acid and sodium
thiosulfate, which is a reducible sulfur source.
Most Salmonella strains can reduce thiosulfate
and produce hydrogen sulfide, which reacts
with the ferric acid in the medium turning it
black. Therefore, Salmonella colonies growing
on SSA will develop black centers. The Shigella
species, in contrast, lacks the enzymes
necessary to reduce sulfur; therefore, it
will maintain its original color.
Microbial growth
In microbiology, growth is defined as an
increase in the number of cells in a population.
The Generation time is the interval it takes for a
population to double in size, either from a
bacteria to two or from 500,000 to a million.
The growth of microorganisms is significantly
affected by environmental conditions, including:
● Temperature
● pH level
● Water availability
● Oxygen
● Nutrient composition
● Pressure
● Radiation
The nutrient content of the medium and the
environmental conditions must be suitable in
order to achieve an efficient growth of
microorganisms.
A nutrient solution called culture medium, or
broth, is used to grow microorganisms in the
laboratory. Every microorganism has dierent
nutritional needs and ability to obtain nutrition
from media through various metabolic
pathways.
Therefore, the amount and type of nutrients to
be added in a medium must be carefully
considered to ensure successful growth and
production of desirable metabolites.
Streaking
One drop of bacterial solution contains millions
of cells. To identify a species, individual
strains have to be grown separately, starting
from a single cell. The most commonly used
method to isolate strain originating from a
single cell is called the streaking method. There
are several different ways to streak a plate.
 immediately heated using the Bunsen burner so
that any air movement is outward.
● The bottle or tube are opened for the
minimum time possible, and while open, all
work is performed close to the Bunsen burner
flame.
● Media and equipment are sterilized to
prevent the growth of unwanted
microorganisms.
Figure 1: Streaking pattern on the agar plate.
The colored lines indicate the path of the wire
loop.

Streaking methods all follow the same basic

principle: a large number of cells are spread
over the surface of an agar plate in a sequential
fashion until the cells are spread far apart
from one another. The numbers and colors
refer to the order of the streaks. For example,
streak number two is started by placing a sterile
loop inside streak number one to pick up
bacteria.
Following streaking, the plate is incubated and
the cells start to divide and form colonies.
Single cells will form colonies of genetically
identical clones. Isolated colonies usually grow
larger than colonies situated closely together
due to less competition for nutrients. These
isolated colonies can be used to make a pure
culture, a liquid or solid culture in which all
bacteria are genetically identical, for further
study. In some cases, the motility or
morphology of the colony can also provide
useful information for identification.
Figures 2 and 3: Left- A contaminated petri dish.
Various species of fungi have grown in this petri
dish. Right- Sterile technique used in material
transfer from the bottle. The neck of the bottle
is heat-sterilized by the Bunsen burner flame.
Logarithmic scale
The logarithmic scale is a nonlinear scale that is
used when measurements differ by several
orders of magnitude.
Have a look at the following three functions
plotted on a linear scale (left) and a logarithmic
scale (right). Notice how the exponential
function becomes a straight line on the
logarithmic scale.

Sterile technique
Sterile technique is used to ensure a "clean" lab
environment. It is essential to ensure the
reliability of experimental results.

Sterile practices are especially important when

working with microorganisms. A single spore or
tiny bacterium can overgrow your whole
medium and destroy your experiment.

The following steps are used to keep laboratory Common uses for a logarithmic scale include
work sterile: earthquake strength, sound loudness, light
● Laboratory doors and windows are kept intensity, growth curves, and pH of solutions.
closed to prevent air currents, preventing
surface microorganisms becoming airborne.
● The wire loop and glass spreader are sterilized
before and after use with a Bunsen burner to
prevent the introduction of unwanted LABSTER
microorganisms.
BACTERIAL ISOLATION:
● Lids from bottles and tubes are held when
removed, and not placed on the bench during Which bacterial species is contained in the
material transfer from one bottle or tube to blood sample from Max?
another. - Salmonella
● The neck of a bottle or tube must be
 o We can identify Salmonella
bacteria on the plate by it’s
black. Salmonella Shigella agar
contains the compounds ferric
acid and sodium thiosulfate.
Most Salmonella strains can
reduce thiosulfate and produce
hydrogen sulfide, which then
reacts with the ferric acid in the
medium turning it black
Which method can we use to identify if the
antibiotic-resistant bacteria in the chicken shed
sample are Salmonella?
- Grow the bacteria on a Salmonella
shigella agar plate
Which statement best describes the purpose of
the T-streaking technique?
- To decrease the concentration of
bacterial cells on the agar surface
Based on the results 0f the streaking method
and previously inoculated plate, how can you
tell that the colony was successfully isolated?
- The bacterial colony is deposited few
millimetres away from the other
colonies on the surface of the agar
plate with uniform, clearly defined
shape and color.

Why are the bacteria samples stored in the

fridge?
- Reduce bacterial growth
o The bacteria will grow much
more slowly if they are kept in
the fridge, giving us more time
to analyze the bacteria.

What conclusion can you draw from the graph?

- The wire loop is not sterile

The flame kills all the microbes and sterilizes the

wire loop. Why is this important to do before
working with the chicken farm sample?
- To avoid contamination
o Since there are bacteria
everywhere, it is very important
to keep all equipment sterile.
Otherwise the contaminating
bacteria from the lab room
might overgrow your agar plate
and we won’t be able to tell
what bacteria came from the
chicken farm.

There are black and red antibiotic-resistant

bacterial species growing on the agar plate. Can
you identify these species?
- Salmonella and E.coli

Why is it important for us to isolate a single

colony of bacteria from the chicken farm
sample?
- We require a sample in which all the
bacteria cells are genetically identical
o The bacterial sample we send
for genetic analysis must
contain cells that are all genetic
clones. We can ensure this by
sending a single colony for
analysis.