EXPERIMENT 7: DIVERSITY

OF BACTERIA
Objective(s)  At the end of this lesson, students should be able to: 
i. To demonstrate Gram staining technique in classifying bacteria
ii. To identify Gram-positive and Gram-negative bacteria
iii. To identify different shapes of bacteria

Gram stain is a widely used method of staining bacteria as an aid to  their
Purpose identification. It was originally devised by Hans Christian  Joachim Gram, a
Danish doctor. Gram stain differentiates two major  cell wall types. Bacterial
species with walls containing small amount  of peptidoglycan and
Part 03
characteristically, lipopolysaccharide, are Gram negative whereas bacteria
with walls containing relatively large  amount of peptidoglycan and no
lipopolysaccharide are Gram-positive.  Apart from Gram staining technique,
the identification of bacteria can  also be based on shapes. The three most
common shapes are spheres,  rods and spirals. 
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A Part 01

Indepedent variable

Depedent Variable

controlled variable
Material & Apparatus
microwave
Hot water
Petri dishes
Beaker
Nutrien agar
Cotton swabs
Clorox wipes
Zipper lock bag
Adult supervision
Procedure

You’ll need a clean, microwave-safe container (a

quart-sized bowl works great) to mix the agar with
water and then boil it. These proportions make
enough nutrient agar to prepare two Petri dishes.
Stir these together well:
• ½-teaspoon agar (about 1.2 grams)
• ¼ cup (60 mL) of hot water
Procedure

Bring this mixture to a boil for three minutes to

completely dissolve the agar. CAUTION: Adult
supervision is required to boil water. If you are
using a microwave oven to boil the mixture, be
careful not to let it boil over. The mixture should be
clear with no particles floating in it after boiling.
Procedure

Remove the mixture from the microwave and allow

it to cool for 3 to 5 minutes before moving on to the
next step.
Procedure

Take the lid off of the Petri dish (the lid is larger
than the dish) and carefully cover the bottom-half
of the Petri dish with warm nutrient agar mixture.
Procedure

5
Loosely cover the bottom portion (set the lid ajar so
excess moisture can escape) and allow the mixture
to cool and harden for at least an hour.

NOTE: Just like gelatin, agar needs to boil for a

certain amount of time to properly gel. If necessary,
pour any unset mixture in each Petri dish back into
the bowl (Cover the empty dishes!) and microwave
it again until you see it boil. Watch it boil (but not
boil over) for 10-15 seconds before turning off the
microwave. There should be no “floaties” in it. Pour
the hot agar mixture back into the dishes (cover
them!) as you did before and it should solidify within
an hour.
Procedure

It’s time to collect some bacteria on the end of a

cotton swab! The classic test is to roll a clean
cotton swab in your mouth and then to lightly draw
a squiggle with it on the gelled agar. However,
many people like to test something even more
gross like the keys on a computer, a cell phone
case, the pump handle of a soap dispenser, or the
TV remote control. Unless someone recently
cleaned the buttons on the remote, you may be
seeing some real goobers in a short time. Dampen
a cotton swab and roll it in your fingers as you pull
it across the surface of your choice.
Procedure

Lift the lid off the Petri dish and LIGHTLY draw a
squiggly line in the agar with the end of the cotton
swab. Roll the swab in your fingers as you draw
the line. Replace the lid and label the dish with the
date and the name of the item you tested.
Procedure

Use a sanitizing wipe to thoroughly clean one of

the surfaces you tested in Step 6, e.g. Cellphone
Procedure

With a clean swab, redo the squiggle test in the

other half of the Petri dish from Step 6 to confirm
your cleaning efforts..
Procedure

10
Before growing anything, some people place each
Petri dish into a separate zipper-lock bag. Place
the upside down dishes into a warm – about 98°F
(37°C) is fine – and totally dark place to grow. In a
closed box on a cable box is a great place. In a
short time, you’ll be greeted by an amazing variety
of bacteria, molds, and fungi. You likely see more
and larger colonies over the next few days. You
shouldn’t see too much growth where the
disinfectants (hand sanitizers) were used. You
might even see a “halo” around each location. This
halo is called the “kill zone.” Measure and compare
the size of the kill zone to determine the
effectiveness of different antibacterial agents
Procedure

11

Goobers like you’re growing will often stink

and make their presence known after a short
time. These are not toys or curiosities you’re
growing. Proper disposal is essential for both
safety and sanitation. Seal all the Petri dishes
into larger zipper-lock plastic bags. You can
add a generous shot of chlorine bleach to the
bag before sealing it to add another level of
destruction.
 Data Collection

Slide / 16
 Discussion
Grain straining is a differenti staibning technique that differentiates bacteria into two group gram postive and gram negative. The procedure is based on the ability of microorganisms to retain colon of the strains used during the gram strain reaction.

Gram negative bacteria are decolorized by the alcohol, long the colour of the primary stain,purple. Grain positive bacteria are not decolorized by alcohol and will remain as purple. After decolorization step, a countersttain is used to impart a pink colour
to the decolorzed gram-negative organisms

There are some precautionnary step for this experiment. One of the precautions is to always use young culture of bacteria because old culture of gram-positive bacteria tend to lose ability to retain the crystal.Do NOT open the dishes once things begin
to grow. You could be culturing some serious goobers and not even know it.

Slide / 17
 Conclusion

Slide / 18
References
Campbell, N.A , Recce ,J.B, Ury, L.A, M.l, rassermen, S.A minorsky, P.V & Jakson
R.B (2016) Biology ( 11th .Ed) Pearson Benjamin Cumming

https://www.stevespanglerscience.com/lab/experiments/growing-
bacteria/