BBTL305Microbiology Laboratory

Experiment 2: Culture techniques: Types of culture media, inoculation,

incubation and isolation of pure colonies.
Aim: A.Reparation of Basic Solid Media and Liquid Media
B. Isolation of Bacteria and Fungi from Different Sources
C.Pure Culture Techniques - Streak, Pour and Spread Plates

Introduction
A nutrient material prepared for the growth of micro-organisms in a laboratory is called a
culture medium. Some bacteria can grow well on just about any culture medium; others
require special media and still others like viruses cannot grow on any synthetic medium
yet developed. The microbes that grow and multiply in or on a culture medium are
referred to as a culture.
Criteria that the culture medium should meet
 It must contain the right nutrients for the particular microorganisms we want to
grow.
 It should also contain sufficient moisture, oxygen and a properly adjusted pH.
 The medium must be initially sterile i.e. it must initially contain no living
microorganism so that the culture will contain only the desired microorganisms.
 Finally the growing culture should be incubated at proper temperature.
For eg: For bacteria temperature is 37o c
For fungi temperature is 28-30o c
The basic requirements of a typical medium normally contains a source of nitrogen,
phosphate, carbon, sulphate, iron, magnesium, sodium, potassium and chlorine ions. These
inorganic chemicals are required for the biosynthesis of various cellular biochemicals and
for the maintenance of transport activities across the cytoplasmic membrane.
 Various metals like zinc, cobalt, and manganese are required as trace elements.
Some growth factors such as vitamins and amino acids, may also be included
 Water forms the major constituent (80%--90%) of any media. It mediates all the
enzymatically controlled chemical reaction that occurs in the cell.
 Distilled water is used in bacteriological media to render it safe from the inhibitory
effect of chemicals
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Media are constantly being developed or being revised for use in the isolation and
identification of bacteria that are of interest to researchers in such areas as food, water and
clinical microbiology.
Media can be classified variously using different criteria viz; chemical composition, physical
state, its utility etc. Based on physical state, the media can be classified into liquid and solid
media.
a) Liquid medium:
It is a medium in which the solidifying agent like agar or silica gel is not added. It is
otherwise known as broth medium. It supports the growth of many common heterotrophs.
Microorganisms grow well in liquid media and hence they are used as enriched media.
Uses:
 It is used for the propagation of large number of organisms, fermentation studies and
various other tests.
 The liquid media is not suitable for the isolation of organism in pure culture and
colony characters cannot be studied.
 Eg: Nutrient broth, Citrate broth.

b) Solid medium:
It is a culture media in which a solidifying agent is added. The solidifying agent is usually
agar, which at concentration of 1.5-2.0% forms firm, transparent gels that are not degraded
by most bacteria. Agar is a polysaccharide (complex) derived from marine algae. It melts at
about the boiling point of water but remains in a liquid state until the temperature drops to
about 40o c. It is not considered as a source of nutrient to the bacteria silica gel is
sometimes used as an inorganic solidifying agent for autotrophic bacteria.
Uses:
 Used for developing surface colonies growth of bacteria and molds.
 Essential to isolate organisms from mixed cultures
 Once the agar has solidified, it can be incubated at temperatures approaching 100o c
before it liquefies; this property is useful when thermophiles are being grown.
 Eg. Nutrient Agar, MRBA
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A) REPARTION OF BASIC SOLID MEDIA AND LIQUID MEDIA

Liquid-growth media containing nutrients (broths) are usually solidified by the addition of
agar. Agar-Agar (often called simply agar) is a complex polysaccharide (carbohydrate)
consisting of 3,6-anhydro-L galactose and D galacto-pyranose, free of nitrogen, produced
from various red algae belonging to Gelidium, Gracilaria, Gigarntina and Pterocladia. It
liquefies on heating to 960C and hardens into a jelly on cooling to 40-450C. Agar can be
replaced with gelatin (10-16%) though this cannot be used with incubation temperatures
exceeding 200C or with proteolytic (protein degrading) microbes.

The solidified medium kept in a Petri dish provides an artificial environment suitable for
the rapid growth of microbes. While in the liquefied state, solid media can be added in test
tubes, which are either allowed to cool and harden in a slanted position producing agar
slants or allowed to harden in the upright position producing agar deep tubes.
Potato dextrose agar (PDA) and Czapek-Dox agar (CDA) are routinely used for the isolation
and maintenance of common fungi. Nutrient agar is the most common media for culturing
& maintenance of bacteria.
Requirements
Constituents of media, 1N HCL, 1N NaOH, pH meter, Distilled water, Hot plate / Heater,
Autoclave, Measuring cylinder, Cotton, Culture tubes, Glass rod.
Note: Precautions
1. Do not pour medium in excess of 2/3 of the capacity of the container used for
autoclaving.
2. Cotton plugs should be kept loose when autoclaving.
3. Do not pour media to Petri plates which are too hot since it produces agar
surface and may lead to culture contamination.
4. Pour medium quickly to avoid contamination by air-spores and close the lid
down as soon as possible.
5. Perform the pouring if medium in a cabinet fitted with UV tubes or room with
filtered air.
6. Pouring is to be performed near the flame or under aseptic conditions.
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7. Medium containing slants or deep tubes are to be stored always at low

temperature in dust free environments.
Procedure
1. Dissolve the media ingredients in 500 ml of distilled water.
2. Heat with agitation to dissolve the constituents.
3. Add 20 g agar, bit by bit to the hot water (960C) to dissolve it.
4. Add more distilled water to make 1 liter.
5. Adjust pH of the medium to 7.0, using a pH meter, by adding either acid or alkali, as
the case may be.
6. Plug the flasks and test tubes containing medium
7. Autoclave at 1210 C, 15 lbs pressure for 15 minutes.
8. Allow the autoclave to cool.
9. Sterilized medium (in flasks or tubes) can be stored at room temperature (250C) in
dust free environment (if to be used within a week), or in a refrigerator (if to be
stored longer) (Photo).
B) PREPARATION OF PLATES

1. Pour the sterilized medium into Petri dishes quickly under aseptic conditions.
2. Allow the medium to gel to produce agar plates.
3. Sterilized medium (in flasks or tubes) can be stored at room temperature (250C) in
dust free environment (if to be used within a week), or in a refrigerator (if to be
stored longer) (Photo).
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Photo: Agar plates preparation

C) PREPARATION OF TUBES

1. Allow the tubes to cool in a slanting position (for agar slants) (Photo) and upright
position (for agar deep tubes) (Photo).
2. Sterilized medium tubes can be stored at room temperature (250C) in dust free
environment (if to be used within a week), or in a refrigerator (if to be stored
longer).

Photo: Agar slant preparation

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B] ISOLATION OF BACTERIA AND FUNGI FROM DIFFERENT SOURCES

Aim: a) To isolate bacteria and fungi from soil (rhizosphere), Water and air by serial
dilution technique.
To isolate bacteria and fungi from soil (rhizosphere) and Water by serial dilution
technique.
Introduction:
Rhizosphere is defined as the region of soil immediately surrounding the roots of a plant
together with the root surfaces. The region provides certain characterstic conditions for
increased occurrence of microflora. It is characterized by greater microbiological diversity
than soil away from plant roots. Increased microflora is attributed to such food provided
by added ploughed off portions of root tissues and root exuadates contains sugars,
aminoacids, vitamins and other growth factors which serve as nutrients for micro organims
in rhizosphere.
Rhizosphere effect is beneficial to plants in two ways, firstly, it helps providing nutrition
to plants, secondly it helps plants in combating root diseases. The term rhizosphere effect
indicates overall influence of plant roots on microorganisms. rhizosphere effects is seen
with bacteria than with actinomycetes, fungi, while with regard to protozoa and algae,
there are only negligible changes. The rhizosphere effect increases with age of plants.
Several factors such as soil, pH, soli type, soil texture, temperature, age and condition of
plant are known to influcence rhizosphere.
Principle:
Soil suspension is obtained by shaking roots soil in sterile water from which subsequent
dilution are made by serial dilution technique. 1 ml of appropriate dilution is placed on
suitable agar medium for enumerating bacteria and fungi. Root free samples from
cultivated plates are used as control of judge the changes in microbial population due to
plant growth.
Requirements:
Soil and Water sample, sterile pipettes and Petriplate, Bunsen Burner etc.
Media - For bacteria: NA, For Fungi: MRBA
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Procedure:
Serial dilution plating of soil and water sample:
1. Soil adhering to roots is collected using spatula. 1gm of sample is added to test tube
containing 9ml of sterile distilled water.
2. 9 ml of sterile water blanks were labelled from 1-6 and sterile petri plates were
labelled 10–1 to 10 –6 dilutions
3. The above dilution, i.e. 1g of sample in 9 ml of sterile water to get 10 –1 dilution
(1:10). The diluted sample was shaken to make a uniform suspension of microorganism.
4. 1 ml of this suspension was transferred to water blank 2, under aseptic conditions to
get (1:100) dilution (10 –2) and it was shaken well
5. 1: 1000 dilution (10 –3) was preferred from 10 –2 by adding 1 ml of 10 –2 test tube.
Further dilutions 10 –4 to 10 –6 are prepared by pipetting 1 ml of suspension into respective
water blanks as above (Photo).
6. Same protocol will be followed for water sample. 1 ml of sample is added to test
tube containing 9ml of sterile distilled water.
Plating
1.1ml aliquots from each dilution were transferred to respective petridishes.
2. Approximately 15 ml of cooled medium (45º c) was added to each petridishes and
the inoculums were mixed by gentle rotation of pertidishes (Pour plate method).
3. Observe all the plates for the appearance of bacterial colonies.
4. The number of colonies on each plate is counted and organisms are identified by
staining.
5. Count the number of colonies in the plates, that have colonies in the 30-300 range,
by placing each plate one by one on the platform of a Quebec Colony Counter.
6. Plates were observed for number and distribution of each dilutions and the result
were recorded.
Observation:
Result:
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Photo: Serial dilution technique

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C] PURE CULTURE TECHNIQUES - STREAK, POUR AND SPREAD PLATES

Aim: Pure culture techniques are for isolation & maintenances of single colony.
Requirements:
24 hr culture of bacterial & fungal organism, Media, Sterile Petri plates and pipettes, Wax
marking pencil and spirit lamp, ethanol
Precautions:
1. Avoid pressing the loop or needle too firmly against the agar surface as this will
damage it.
2. Inoculating loop should be cooled by touching the agar surface away from the set of
streaks, before streaking of the inoculums.
3. Petri plate lid should never be lifted completely.
4. Plating of the medium should be done twenty-four hours in advance of performing
the exercise.
5. Use a fresh sterile pipette for each dilution.
6. The medium to be poured in the Petri plates should have a temperature of 45 0C
while transferring a suspension from one tube to another it should be in motion for
the uniform distribution of cells.
7. The plates should be incubated in an inverted position to prevent collection of
condensation on the agar surface. Unless the surface is dry it will be difficult to
obtain discrete surface colonies.
POUR PLATE METHOD
The fore runner of the present pour-plate method was developed in the laboratory of the
famous bacteriologist, Robert Koch.
In this technique, successive dilutions of the inoculum (serially diluting the original
specimen) are added into sterile Petri plates to which is poured melted and cooled (42-450
c) agar medium and thoroughly mixed by rotating the plates which is then allowed to
solidify. After incubation, the plates are examined for the presence of individual colonies
growing throughout the medium.
Pour plates are also used as a means of determining the number of viable organisms in a
liquid such as water, milk, urine or broth culture as well as to determine the hemolytic
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activity of deep colonies of some bacteria, such as the Streptococci, by using an agar
medium containing blood (Photo).
Procedure:
1. Take a sterilized petriplates & label it.
2. Place 0.1ml inoculum in petri plate & pour the medium into plate and rotate the
plate gently to ensure uniform distribution of cells in the medium.
3. Allow the medium to solidify.
4. Incubate the inoculated plates for 24-48 hours at 370 c in an inverted position (lid
on bottom)
SPREAD PLATE METHOD
The spread-plate technique is used for the separation of a dilute, mixed population of
microorganisms so that individual colonies can be isolated. In this technique
microorganisms are spread over the solidified agar medium with a sterile L-shaped glass
rod while the Petri dish is spun on a turntable. The theory behind the technique is that as
the Petri dish spuns, at some stage, single cells will be deposited with the bent galss rod. On
to the agar surface. Some of these cells will be separated from each other by a distance
sufficient to allow the colonies that develop to be free from each other (Photo).
Procedure:
1. Label one nutrient agar plate with inoculum.
2. Take 95 per cent alcohol into a beaker and dip the bent glass rod in it.
3. Aseptically transfer a loopful culture of inoculum in the center of appropriately
labeled nutrient agar plate.
4. Place the inoculated plate on the turntable.
5. Remove the glass rod from the beaker and sterile the bent potion in the Bunsen
burner flame.
6. Cool the rod for 10-15 seconds.
7. Remove the cover of Petri dish and spin the turntable
8. Lightly touch the sterile bent rod to the agar surface and move it back and forth
while the turntable is spinning for spreading the culture over the agar surface.
9. Replace the Petri dish cover when the turntable stops spinning
10. Immerse the bent rod in alcohol and reflame to sterilize it.
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11. Incubate a plates in an inverted position at 370 C for 24 to 48 hrs.

Photo: Pour plate method and Spread plate method

STREAK PLATE METHOD
The streak-plate technique is used for the separation of a dilute, mixed population of
microorganisms so that individual colonies can be isolated. In this technique
microorganisms are streak over the solidified agar medium either in parallel or zigzag
method (Photo).
Procedure:
1. Label all the plates, on the bottom, with the name of the organism(s) to be
inoculated, with a wax marking pencil.
2. Hold the tube containing the broth in the left hand.
3. Sterilize the loop holding in the right hand, remove the cotton wool plug, using the
little finger of the right hand and immediately flame the mouth of the tube.
4. Introduce the loop into the broth and withdraw one loopful of culture.
5. Flame the mouth of the tube, replace the cotton wool plug and place the tube in the
test tube rack.
6. Lift the Petri plate cover the left hand and hold it at an angle of 60.
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7. Place the inoculum (the loop containing the droplet of broth) on the agar surface at
the edge farthest from you and steak the inoculum from side to side in parallel lines
across the surface of area.
8. Reflame and cool the loop and turn the Petri plate to 90. Touch the loop to a corner
of the culture in area 1 and streak the inoculum across the agar in area 2 as above
(The loop should never enter area 1 again).
9. The rest of the agar surface is now used to complete the streaking
10. Replace the lid of the Petri plate, after completing the streaking, and sterilize the
loop by framing.
11. Prepare a streak-plate inoculation of the rest two test cultures.
12. Incubate all the plates at 35 c, in an inverted position, for 48-72 hrs.
Results: