New Mexico State University Virtual Labs Gram Staining Storyboard v1
Joan, if you could give us feedback for the following:
 How is it overall
 Did we get the procedure correct?
 Did we miss any key points?
 Is there anything we should add or remove?
 Text review, are there any information that is incorrect?
 Does the whey separation mean the yogurt is bad. What about the yogurt I opened
this morning that had the water separating?
 Are there different situations where gram staining is used to identify bacteria instead
of growing colonies on a petridish/petrifilm. When would you use gram staining and
when petridish/petrifilm?
 For the slide, how can we tell which side the samples goes on and which side do we
label?
 For the inoculation loop, how much is a small amount? Do we just use a loop full?
 After Safronine, we do not have to repeat with the Gram iodine and alcohol?
 Bibulous paper doesn’t contaminate the slide? Can we use paper that has been used
before or do we need to find sheets that has not been used before
 Is there a specific bacteria we should contaminate the yogurt with. E. coli?
 Can we identify bacteria based on Gram staining and the image under the
microscope? No further testing is necessary?
 Labels for the left side navigation.
1
Hi, lab assistant! Welcome to the
yogurt section of the dairy processing
plant. It appears that our latest batch
of yogurt might have been
contaminated by some harmful
bacteria.
1
New Mexico State University Virtual Labs Gram Staining Storyboard v1
All yogurt has bacteria in it. That is
because yogurt is made by adding a
certain kind of bacteria to milk. These
bacteria are harmless and beneficial to
humans.
However, sometimes, harmful bacteria
like Salmonella or E. coli end up in
yogurt as well, which is a problem.
These bacteria can make people very
sick.
More information:
Salmonella and E.coli
2
New Mexico State University Virtual Labs Gram Staining Storyboard v1
This is what normal, healthy yogurt
looks like. Notice the uniform texture
and color.
Photo of good yogurt.
Now, compare that to this yogurt. Look
at how the whey and the water are
separating. Also, it looks like there are
some air bubbles forming near the
bottom. These are signs of
contamination.
More Info:
Why are air bubbles a sign of
contamination
Photo of bad yogurt
3
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Because we cannot tell if it is
contaminated just looking at it, we will
follow up with testing.
We need to collect yogurt samples and
run some tests to identify if the yogurt
is contaminated with harmful bacteria.
We are going to use a process called
Gram staining developed by Dr.Gram.
It is a process where the bacteria are
stained to identify the bacteria into
two major groups and allows us to
identify harmful and harmless
bacteria.
,, More Info:
Short background about Gram staining
4
New Mexico State University Virtual Labs Gram Staining Storyboard v1
With this process bacteria that are
harmless usually stain purple and are
grouped as Gram-positive. Bacteria
that are harmful stain pink and are
grouped as Gram-negitive. Once we
have stained a sample, we will view
the results under a microscope.
More Info:
What makes some bacteria purple and
others pink
Bacteria that stain purple are Gram
positive and are generally harmless to
people.
Photo of actual Gram positive and
negative slides. Labeled with type of
bacteria
5
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Bacteria that stain pink are Gram
negative and are generally harmful to
people.
We are going to Gram stain samples of
the yogurt. With Gram Staining we can
identify the kind of bacteria present.
The bacteria used to make yogurt are
all Gram positive and harmless, so if
any Gram negative bacteria show up in
this yogurt we know it's contaminated.
6
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Alright, let's start preparing our slides
for Gram staining!
To prevent bacteria from
contaminating our work area, we have
wiped down the table with a 70%
alcohol solution. We will also light a
bunsen burner. This will kill any
microorganisms in the air around our
work area.
Bring the match to the bunsen burner
to light it.
7
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Use a permanent marker to draw a
circle on the slide. The sample will go
on the opposite side of the marking.
Hold down the mouse button and drag
the pen to write your initials on the
end of the slide, so if anyone has
questions about the slide, they know
who to talk to.
8
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Now we are going to prepare a sample
of the yogurt. We will add a diluent to
dilute the sample and make it easier to
work with by adding liquid volume.
We will use a diluent of Phosphate
Based Saline to thin out the mixture so
we can see through it in the
microscope.
More info:
What is phosphate Based saline?
Start by placing 1 mL of Phosphate
Based Saline diluent into a test tube
using a pipette.
More info:
What is pipette?
9
New Mexico State University Virtual Labs Gram Staining Storyboard v1
This is an inoculation loop, which is a
small loop of wire used to collect
single colonies or drops of
contaminated substances. We'll use an
inoculation loop to place a small
amount of yogurt into the diluent.
Photo of the inoculation loop
Start by sterilizing the wire loop in the
flame of the Bunsen burner. Hold it
directly in the flame until it glows red
hot.
Photo of sterilizing the loop
10
New Mexico State University Virtual Labs Gram Staining Storyboard v1
After it has cooled down, use the loop
to pick a small glob of yogurt from the
contaminated sample. Make sure to
grab the whey, the solid part.
Place the yogurt sample into the
diluent.
11
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Now shake the test tube for 30
seconds to mix up the sample.
Using a pipette, pick up a small
amount and place a few drops in the
middle of your prepared slide. If you
are using disposable pipettes, be sure
to use a fresh one, you don't want your
solution contaminated with
substances left over from the previous
solution..
Photo of the sample on the slide.
12
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Now we will heat fix the slide. This
prevents the bacteria from being
washed away when we begin staining
process. Drag the slide over the
burner, but be careful not to put it
directly in the flame! We don't want to
burn the sample, just dry it out so it
won't wash off.
Now we begin the staining process.
After the staining is complete, these
slides will be ready to look at under a
microscope.
13
New Mexico State University Virtual Labs Gram Staining Storyboard v1
We use 4 chemicals to stain a slide. We
put them on in a specific order, and
rinse the slide off under running water
in between each chemical.
The first chemical is Crystal Violet.
This is our purple stain, which will
indicate Gram positive bacteria.
More info:
What is Crystal violet
14
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Grab the eye dropper and squeeze
enough drops to cover your sample.
Let that sit for 1 minute. Now rinse it
under running water.
Photo: of crystal violet slide.
15
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Now, grab the jar of Gram's Iodine.
Gram's iodine is a fixer. It will fix the
dye to the cell walls of the bacteria,
and make it permanent.
More info:
What is Gram's iodine?
Grab the eyedropper and squeeze
several drops onto the sample, enough
to cover it completely.
photo of: Gram's iodine slide
16
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Let that sit for 1 minute. Now rinse it
under running water.
Now, we are going to use the alcohol
solution. This will wash off the excess
dye, leaving only the dye fixed to the
bacteria.
more info: what is the 95% alcohol
solution?
17
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Grab the alcohol eye dropper and
cover the sample in alcohol.
photo: alcohol on slide
Let the alcohol sit for 30 seconds. Time
is very important for this step, don't
let the alcohol sit for more than 30
seconds.
Now rinse it off under running water.
.
More Info
What happens if alcohol is left for
more than 30 seconds.
18
New Mexico State University Virtual Labs Gram Staining Storyboard v1
For the final step, grab the bottle
labeled Safronine. This is the pink dye,
and will stain any remaining undyed
bacteria (the Gram negative ones)
pink.
MOre info: What is safronine?
Cover the slide with safronine.
Photo: of safronine slide.
19
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Let that sit for 1 minute. Now rinse it
under running water.
Now, our slide is ready, but it is still
wet. Place it between two sheets of
bibulous paper, which is highly
absorbent paper used for drying off
slides, especially those containing
organic material.
20
New Mexico State University Virtual Labs Gram Staining Storyboard v1
Push the paper down a few times to
blot the water off the slide.
Good job, you've got one slide done.
Now see if you can do the next one on
your own. You can always click on the
hint button if you need some help
remembering which step comes next.
Photo of the slide completed.
21
New Mexico State University Virtual Labs Gram Staining Storyboard v1
,
…
(Own their own section)
Storyboard Notes: Our plan here is to
allow the user to go through the
motions on their own, with hints
available if they forget.
Congratulations! Here's what we found
under the microscope. The image on
the left shows no sign of Gram
negative and is healthy yogurt. The
one on the right is contaminated.
Notice the pink stained bacteria. Those
are Gram negative, and harmful to
humans.
Photo of what the bacteria look like
under the microscope.
22
New Mexico State University Virtual Labs Gram Staining Storyboard v1
.,
Look at all those Gram negative
bacteria, it's a good thing we caught
this before the product shipped.
23