TO STUDY THE RATE

OF BACTERIA PRESENT
IN AGAR-AGAR GEL

INDEX

S.
PAGE NO
NO TOPIC
1. AGAR 1
2. TYPES OF AGAR IN MICROBIOLOGY 2
3. COMPOSTION OF AGAR AGAR 3
4. PRODUCTION OF AGAR 4
5. AGROSE 5
6. EXPERIMENT 6-7
 7. BIBLOGRAPHY 8

AGAR

Agar or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the

cell walls of some species of red algae, primarily from ogonori (Gracilaria) and "tengusa"
(Gelidiaceae) As found in nature, agar is a mixture of two components, the
linear polysaccharide agarose and a heterogeneous mixture of smaller molecules
called agaropectin. It forms the supporting structure in the cell walls of certain species of
algae and is released on boiling. These algae are known as agarophytes, belonging to
the Rhodophyta (red algae) phylum. The processing of food-grade agar removes the
agaropectin, and the commercial product is essentially pure agarose.
Agar has been used as an ingredient in desserts throughout Asia and also as a
solid substrate to contain culture media for microbiological work. Agar can be used as
a laxative; an appetite suppressant; a vegan substitute for gelatin; a thickener for soups;
in fruit preserves, ice cream, and other desserts; as a clarifying agent in brewing; and
for sizing paper and fabrics.

TYPES OF AGAR IN MICROBOLOGY

Different algae produce various types of agar. Each agar has unique properties that suit different
purposes. Because of the agarose component, the agar solidifies. When heated, agarose has
the potential to melt and then solidify. Because of this property, they are referred to as "physical
gels." Polyacrylamide polymerization is an irreversible process, and the resulting products are
known as chemical gels.
Given below is a list of different types of agar that support the different strains of bacterial growth:

1. Blood Agar: A bacterial growth media is blood agar. It is mostly used to cultivate
pathogenic bacteria such as Streptococci.
 2. Luria Bertani (LB) Agar: are nutrient-rich plates used for bacterial growth. They are
frequently utilized in cloning operations to increase the number of antibiotic-resistant,
competent microorganisms. The same ingredients, without the agar, can be used to
make liquid LB medium.[25]
3. Chocolate Agar : Chocolate Agar (CAP or CHOC) is a nonselective improved medium
used to detect and isolate finicky pathogens.Chocolate agar is created by heating blood
agar, which subsequently splits the red blood cell (RBC), releasing nutrients that help in
the development of fastidious bacteria such as Haemophilus and Neisseria
species..Chocolate agar is very identical to blood agar, except that the red blood cells
are destroyed during the manufacturing process when they are introduced to the molten
agar foundation. This causes cell lysis,
4. MacConkey Agar: Alfred Theodore MacConkey produced the first solid differential
medium, (MAC), in the twentieth century. MacConkey agar is a selective and differential
media used for the isolation and differentiation of non-fastidious gram-negative rods,
notably members of the Enterobacteriaceae and Pseudomonas genera. MacConkey
Agar Applications: Gram-negative intestinal bacteria are isolated using MacConkey agar.
It is used to distinguish lactose fermenting gram-negative bacteria from lactose non-
fermenting gram-negative bacteria. It's used to isolate coliforms and intestinal pathogens
from water, dairy products, and biological samples [27]
5. Nutrient Agar: Nutrient Agar is a basic culture medium that is often used for the
cultivation of non-fastidious microorganisms, as well as for quality control and purity
checking prior to biochemical or serological testing. Many bacteria that are not
particularly fastidious have been grown and counted on nutrient agar .By adding different
biological fluids such as horse or sheep blood, serum, egg yolk, and so on, the media
can be made suitable for the cultivation of other fastidious organisms. [28]
6. Neomycin Agar: Neomycin blood agar is a popular selective medium for isolating
vancomycin-resistant enterococci from feces; however, not all isolates are recovered
using this medium, perhaps due to excessive neomycin concentrations. [29]

COMPOSITION OF AGAR AGAR

Agar consists of a mixture of two polysaccharides: agarose and agaropectin, with agarose
making up about 70% of the mixture. [19] Agarose is a linear polymer, made up of repeating units
of agarobiose, a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose.
[20]
 Agaropectin is a heterogeneous mixture of smaller molecules that occur in lesser amounts,
and is made up of alternating units of D-galactose and L-galactose heavily modified with acidic
side-groups, such as sulfate and pyruvate.

Agar exhibits hysteresis, solidifying at about 32–40 °C (305–313 K, 90–104 °F) but melting at

85 °C (358  K, 185 °F).[22] This property lends a suitable balance between easy melting and good
gel stability at relatively high temperatures. Since many scientific applications require incubation
at temperatures close to human body temperature (37 °C), agar is more appropriate than other
solidifying agents that melt at this temperature, such as gelatin.
 PRODUCTION OF AGAR AGAR
For the hot-water extraction, Gelidium is more resistant and extraction under pressure
(105-110°C for 2-4 hours) is faster and gives higher yields. Gracilaria is usually treated
with water at 95-100°C for 2-4 hours. The remainder of the process is the same for both
types of raw material. The hot extract is given a coarse filtration to remove the seaweed
residue, filter aid is added and the extract is pumped through a filter press equipped with
a fine filter cloth. The extract is thick and will gel if allowed to cool, so it must be kept
hot during the filtration processes.

The filtrate is now cooled to form a gel, which is broken into pieces (Figures 7 and 8).
This gel contains about 1 percent agar. The remaining 99 percent is water that may
contain salts, colouring matter and soluble carbohydrates. The gel may be treated with
bleach to reduce any colour, washed to remove the bleach, and allowed to soak in water
so that most of the salts can be removed by osmosis. The wash waters are drained and
the remainder of the process is concerned with the removal of the 99 percent water in
the gel. Either of two methods can be used for this.

The original method of water removal is the freeze-thaw process. The gel is slowly frozen
so that large ice crystals form. The structure of the gel is broken down by the freezing so
that when the material is thawed most of the water drains away, leaving a concentrated
gel that now contains about 10-12 percent agar (this means about 90 percent of the
original water content has been removed, and with it went a high proportion of any salts,
soluble carbohydrates and soluble proteins that may have been present in the gel).
Sometimes this gel is placed between porous filter cloths and squeezed in a hydraulic
press to remove more water. However, this is a slow process, and usually the thawed
material is simply drained and placed in a hot-air dryer. After drying it is milled to the
required particle size, usually about 80-100 mesh size. Because of the refrigeration
costs, this freeze-thaw process is relatively expensive, compared to the alternative
described next.
 AGROSE
Agar can be divided into two principal components: agarose and agaropectin. Agarose is
the gelling component; agaropectin has only a low gelling ability. There are several
methods of producing agarose; many rely on removing the agaropectin from the agar.
There are only a small number of processors who produce purified, high quality agarose
for a small but growing market, mainly in biotechnology applications. These processors
 use good quality agar as their starting material rather than seaweed, and are often not
in the seaweed processing business. Armisen and Galatas (1987) summarize the
methods that have been used to isolate agarose from agar, and discuss the
specifications expected for a high quality agarose.

EXPERIMENT
1. To get started, you’ll need to make your agar and fill some petri dishes. Use a
clean, microwave-safe container to mix the agar with water and then boil it. A
quart-sized bowl works great. These proportions make enough nutrient agar to
prepare two petri dishes. Stir these together well:

 ½-teaspoon agar (1 1/5 grams)

 ¼ cup (60 milliliters) of hot water


 2. Bring the agar 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.

3. Remove the mixture from the microwave or the stovetop and allow it to cool for
three to five minutes before moving on to the next step.
4. Take the lid off of the petri dish and carefully cover the bottom half of the petri
dish with the warm nutrient agar mixture.

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

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 and
microwave it again until you see it boil. Watch it boil for 10-15 seconds before turning
off the microwave, but don’t let it boil over. Make sure there are no floating particles.
Pour the hot agar mixture back into the dishes just like before and it should solidify
within an hour.
6. Once the agar is set and cooled, it’s time to collect some bacteria on the end
of a cotton swab. Roll a clean cotton swab in your mouth or test something
even more gross, like the keys on a computer, a cell phone case, the pump
handle of a soap dispenser or the remote control.

NOTE: You might want to collect a sample from a computer keyboard on half of the
Petri dish and leave the other half for the test in Steps 8 and 9. Remember, you must
use clean cotton swabs for each sample. In order to get a good sample, lightly
dampen the cotton swab with water. Be sure to roll the swab on the surface you’re
testing so that it’s completely covered and has picked up as much bacteria as possible.
You want to cover the entire cotton end of the swab with invisible bacteria.

7. 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.

NOTE: Moisture coming from the agar can be a problem. Many people turn the petri
dish over during this time to prevent moisture from dripping onto the growing
colonies.
SUGGESTION: Place no more than a drop of a hand sanitizing gel in the middle of
one of the squiggles. The hypothesis is that the antibacterial chemical in hand sanitizer
will keep bacteria from growing there.

8. Use a sanitizing wipe to thoroughly clean one of the surfaces you tested in
Step 6.
9. With a clean swab, redo the squiggle test in the other half of the Petri dish from
Step 6 to confirm your cleaning efforts.
10. Before growing anything, some people place each Petri dish into separate
zipper-lock bags. Place the upside down dishes into a warm, dark place to
grow. A temperature around 98 degrees Fahrenheit (37 degrees Celsius) is
best, like in a closed container on top of a cable box. 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 hand sanitizer was used — you might even see a halo
around each location, which is called the kill zone. Measure and compare the
size of the kill zone to determine the effectiveness of different antibacterial
agents.

REMEMBER: Do not open the dishes once things begin to grow. You could be
culturing some serious bacteria and not even know it. The comfort is that they were
around you all the time anyway and now you can see them. Just be careful.

11. Bacteria, like what 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 in larger zipper-lock plastic bags. You can add a generous shot of
chlorine bleach to the bag before sealing for an extra level of destruction.

 REMEMBER: When you’re done, do not open the bags. After you’ve analyzed the
cultures, dispose of the sealed bag containing the petri dishes with bacteria into the trash.
At the end of the growing period, observe the petri dishes and write down, draw or
photograph what you see. Turns out, Mom is right — it is super important to wash
your hands with soap and warm water whenever you can.

HOW DOES IT WORK

You’re likely to have a huge variety of colors, shapes and smells in your tiny
worlds. Count the number of colonies on the plate, note the differences in color,
shape and other observable properties. Getting bacteria to grow can be a little
tricky, so don’t get discouraged if you have to make more than one attempt. Allow
enough time for them to grow, too. You need millions of them in one place just to
see them at all. They’re that tiny. In a lab, you’d use your trusty inoculating loop to
pick up a bit of the bacteria in order to create a slide for further study under a
microscope. Compare the number, color and size of colonies grown from different
test locations. Which part of your home has the most bacteria? Which one
surprised you the most?
 BIBLOGARPHY

https://www.stevespanglerscience.com/
lab/experiments/growing-bacteria/
https://www.fao.org/3/y4765e/
y4765e06.htm
https://en.wikipedia.org/wiki/Agar