EXPERIMENT – 5

Aim:
Demonstration of presence of microflora in the environment by exposing nutrient agar
plates to air.

Materials Required:

 Beef extract
 Water
 Agar (marine algae)
 Peptones
 Petri dishes

Theory:

Microbes are minute, unicellular organisms that are invisible to the naked eye. There are
different types of microbes like fungi , bacteria , viruses, archae etc. Microbes are found
everywhere in our environment : in air, soil, water surface and almost every surface you can
think of . There are mainly 2 types of microbes :

1. Non-Pathogenic microbes: These microbes are incapable of causing disease, i.e. they are
harmless to other organisms. These type of microbes are useful to man as they are
involved in:
 Manufacturing foods such as butter, cheese
 Alcohol, lactic acid
 Probiotics and fermented products
 Antibiotics
 Gradual decomposition of organic matter (dead animals, plants and their waste)
on or in the soil.
 Bio-fertilizers etc.

Some non-pathogenic microbes live on the surface of animals as normal flora.

2. Pathogenic microbes: these are the microbes that can cause disease. Pathogenecity is the
capacity of a microbe to cause a disease and thus they are harmful to other organism . the
diseases caused by pathogenic microbes are called infections. The agents that cause
disease fal into 5 groups: bacteria,fungi,protozoa,viruses, and helminthes (worms).
Microbes can enter the body through the four sites listed below:

 Respiratory tract (mouth and nose). This can occur while talking sneezing , coughin etc
via air through water droplets. e.g. influenza virus which causes the flu.
 Gastrointestinal tract (mouth oral cavity) e.g. Vibrio cholerae which causes cholera.
 Urogenital tract e.g. Escherichia coli which causes cystitis.
 Breaks in the skin surface e.g. Clostridium tetani which causes tetanus.

There can be different air through which these microbes and travel and spread. Air (outdoor)
i.e the dust practicles leads to sticking of the microbes and thus leading to its spread . On the
other hand indoor air i.e. the air that is spread by living organisms (humans ) can also be
responsible for spreading microbes . This occurs when an infected person sneezes and
coughs and release the microbes into the air , which might land on another person thus
infecting them.
 Passive exposure and active exposure:

 Passive exposure : When petri- plates are kept out in the environment for the
microbes to enter the nutrient medium is known as passive exposure.

 Active exposure: When petri -plates are are not kept in open air but the air pumped
into it to support the growth of microbes is called active exposure.

Organisms varying due to temperature:

Air has a variety of organisms which vary with temperature as well.

 Psychrophiles:

Bacteria that grow at temperatures in the range of -5oC to 30oC, with optimum temperatures
between 10oC and 20oC, are called psychrophiles. These microbes have enzymes that catalyze
best when the conditions are cold, and have cell membranes that remain fluid at these lower
temperatures.

 Mesophiles:

Microbes that grow at optimal temperatures in the range 20 oC to 40oC, are called mesophilic.
Important members of this group are those that live in and on warm blooded creatures, such as
humans. Pathogenic bacteria and included here, as are symbiotic bacteria that live in the human
body without harming it.

 Thermophiles:

Certain bacteria can live and grow at temperatures that exceed 50oC. These
are thermophilic microbes that can tolerate the very harsh conditions decomposing organic
material, like the hot springs. or deep in the oceans by thermal vents bubbling up from the hot
rocks just below the earth's crust.
Preparation of nutrient agar:

1)Dissolve the following ingredients:

 Beef extract - 3.0 gm

 Peptones - 5.0 gm (meat, casein, gelatin)
 Water -1 litre

2)To this add 15 grams of agar . the ph should be adjusted to 6.8.

3) Make sure the agar is made of marine algae which solidifies at 45 oC and melts at 95 oC.

4) Once the nutrient media is prepared :

 Sterilize agar- This is done by autoclaving 15 lbs pressure (121°C) for 15 minutes.
 Pour the plates- Each petri-plate (90mm) should contain 2o ml of the media.
 Label the plates ( according to which environment are they exposed to )

5) The plates with the nutrient medium are then exposed to different environment for the growth
of different types of bacteria . These plates can be exposed to outdoor air, indoor air ,or any
objects which might contain microbes. Some of the plates are left unexposed for comparison.

6) Seal the plates with paraffin wax , invert them and keep them in an incubator. Inoculation can
be passive (simply opening wich will make the particles to set up on the petri-plate) or active
(directing air from certain area in media).

7) Study the growth obtained based on temperature differences.

Observations: (Expected outcome)

1)Maximum growth of the microbes were found to be at a high temperature i.e. at 40 °C.

2)Staphylococcus, clostridium and bacillus are found in air.

Staphylococcus Bacillus

Clostridium