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Quantitative Estimation of Microrganism in Soil

The document discusses qualitative and quantitative estimation of microorganisms in soil, highlighting their importance in soil health, environmental monitoring, and agriculture. It details methods such as the Plate Count Method for isolating microbial colonies and the Most Probable Number (MPN) Test for assessing water quality. The procedures for both methods are outlined, emphasizing the significance of microbial populations in understanding soil dynamics and water safety.

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Siddharth Nair
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14 views

Quantitative Estimation of Microrganism in Soil

The document discusses qualitative and quantitative estimation of microorganisms in soil, highlighting their importance in soil health, environmental monitoring, and agriculture. It details methods such as the Plate Count Method for isolating microbial colonies and the Most Probable Number (MPN) Test for assessing water quality. The procedures for both methods are outlined, emphasizing the significance of microbial populations in understanding soil dynamics and water safety.

Uploaded by

Siddharth Nair
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Qualitative Estimation of

Microorganism from Soil

Siddharth Nair
Msc Microbiology
Sir Syed Institue of Technical
Studies,Taliparamba
Introduction
 A qualitative estimation of microorganisms from soil involves observing and describing the presence of
different types of microbes within a soil sample using techniques like microscopic examination, where
you can identify the morphology and characteristics of bacteria, fungi, and other microorganisms
without necessarily counting their exact numbers, essentially giving you a general idea ofSoil is a
complex and dynamic environment that hosts a vast diversity of microorganisms, including bacteria,
fungi, archaea, actinomycetes, and viruses. These microbes play critical roles in soil health, nutrient
cycling, plant growth, and ecosystem functioning. The quantitative estimation of microbes in soil is
essential for understanding these roles and the overall microbial dynamics within the soil environment.
Accurate measurements of microbial populations can provide insights into soil fertility, microbial
biodiversity, and the impacts of agricultural practices, climate change, and pollution on soil ecosystems.
 Quantitative microbial estimation is important for several reasons:
 Soil Health Assessment: Microbial populations directly influence soil fertility and plant growth through
processes such as nutrient cycling, organic matter decomposition, and disease suppression.
 Environmental Monitoring: Estimating microbial populations helps in monitoring the effects of
environmental disturbances, such as pollution or land-use changes, on soil microbial communities.
 Agriculture: Understanding microbial communities helps in managing soil microbiomes for enhanced
crop productivity, disease resistance, and sustainable agricultural practices.
 Bioremediation: Microbial populations are used in soil to degrade pollutants; quantitative assessments
help monitor and optimize these processes. the microbial diversity present in the soil.
Plate Count Method
 Plate count method is the most frequently utilized technique. This method uses repeated
dilution and colony forming unit (CFU) counts to isolate microbial colonies. With this method, the liquid
sample is added to the petri dish before the agar medium solidifies (this method is called the
pour plate method). colonies form inside and on top of the surface, After the medium solidification. The
confluent colonies, on the other hand, are the ones that are growing inside the medium.
Objective of the Plate count Technique?
 To separate the microorganisms that are in suspension.
 Colony forming units (CFU) per milliliter can be used to estimate how much viable microbial population
is present.
 From a mixed population, to separate the purest cultures of microorganisms
 to separate bacteria from several microorganisms to study the traits of colonies.
Principle
 The Plate count method is based on the idea that each viable bacterium will proliferate and form a
distinct colony when an agar media containing microorganisms is incubated. This procedure involves
properly mixing a particular volume, often 1 mL, of the serially diluted liquid sample with around 15 mL
of molten agar medium at a temperature of 40–45°C (less than 50°C) in a Petri plate.
 Allow the medium to solidify, then it is normally incubated for 24 to 48 hours at 37°C. After incubation,
the sample’s viable microorganisms will form separate colonies both inside and on the surface of the
medium. The visible colonies can be counted, and the following formula can be used to determine
CFU/mL
• Procedure
Write 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, and 10-7 in the dilution boxes.
• Prepare the first dilution by putting 1 ml or 1 g of the sample into a 9 ml dilution blank marked 10-1.
This dilutes the original sample 10 times
• To make sure the organisms are spread out evenly, mix them well (cells).
• From the first dilution, move 1 ml of the suspension while in motion to the dilution blank 10 -2 with a
clean and sterile 1 ml pipette. This will dilute the original specimen/suspension 100 times (1/100 or
10-2).
• With a new, clean pipette, move 1 ml of the 10-2 suspension to the 10-3 dilution blank. This makes
the original sample 1000 times weaker (10-3).
• Use a new, clean, sterile pipette each time until the original sample has been diluted 10 –7 times.
• With the right pipettes, add 1ml or 0.1ml of suspension from the right dilutions (10 -1 to 10-7) to
sterile Petri dishes while moving. For each dilution, you need to use 2 or 3 Petri dishes.
• Melt the PCA medium and let it cool to 45°C. Add about 15 ml of the melted medium to each Petri
plate with the diluted sample. Turn each plate gently to mix the contents and spread the cells
throughout the medium.
• Let the plates settle down.
• At 37°C, Incubate these plates upside down for 24 to 48 hours
Most Probable Number (MPN) Test for Water Quality

 Most Probable Number (MPN) Test for Water Quality


 The most probable number (MPN) test is a statistical method test based on the random
dispersion of microorganisms per volume in a given sample.
 In this method, measured volumes of water are added to a series of tubes containing a liquid indicator
growth medium.
 The media receiving one or more indicator bacteria show growth and a characteristic color change. The
color change is absent in those receiving only an inoculum of water without indicator bacteria.
 From the number and distribution of positive and negative reactions, the MPN of indicator organisms in
the sample may be estimated by reference to statistical tables.
 MPN test is completed in three steps:
 Presumptive test
 Confirmed test
 Completed test
Continuation....
 Objectives of MPN Test
 To enumerate the number of bacteria present in the drinking water by the MPN method.
 To identify the bacteria present in the drinking water sample.
Procedure of MPN Test
I. Presumptive Test
 Prepare MacConkey purple media of single and double strength in test tubes with Durham’s tube and autoclave
it.
 Take three sets of test tubes containing five tubes in each set; one set with 10 ml of double strength (DS) other
two containing 10 ml of single strength (SS) .
 Using sterile pipettes, transfer 10 ml of water to each of the DS broth tubes. Transfer 1 ml of water sample to
each of 5 tubes of one set of SS broth and transfer 0.1 ml water to five tubes of remaining last set of SS broth
tubes.
 Incubate the tubes at 37°C for 24 hours.
 After incubation, observe the gas production in Durham’s tube and the color change of the media.
 Record the number of positive results from each set and compare with the standard chart to give presumptive
coliform count per 100 ml water sample.

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