Isolation of microbial strain from natural geographical region

Microorganisms are generally found in nature (air, soil, and water) as mixed
populations. The process of obtaining a pure culture by separating one species of microbe
from a mixture of other species is known as the isolation of the organisms. The primary
culture from a natural source is usually a mixed culture containing microbes of different
kinds. However, in the laboratory, the various species may be isolated from one another. A
culture that contains just one species of microorganism is called a pure culture. A culture is a
population of microorganisms grown under well-defined conditions. A mixed culture contains
a very small number of particular species of microbe in comparison to the total number of
microorganisms, such culture is called mixed culture. A culture containing only one species
of microbe is called pure culture.
Here are some methods for collecting soil samples for microbiological analysis:
 Remove surface litter: Remove any dead plant material or surface litter.
 Use the right equipment: Use a shovel, auger, trowel, or other suitable tool to collect the
soil. Push probes, hammer probes, and bucket augers are commonly used.
 Collect from the right area: Collect samples from the surface to the tillage depth, which is
usually about 6 inches.
 Collect multiple samples: Collect equal volumes of soil from random points within the
same horizon.
 Combine samples: Mix all the collected samples in a clean plastic bucket or tray. Break up
any clods or lumps before mixing.
 Remove unwanted material: Remove any material other than soil, such as stones, pebbles,
or roots.
 Store samples properly: Place the desired quantity of the composite sample into a labeled
plastic bag. Refrigerate samples and ensure the temperature does not exceed 7°C when they
arrive at the laboratory. Analyze the samples within 24 hours of sampling.
 Collect samples aseptically: Wear a clean lab coat, clean gloves, and a hair net to avoid
contaminating the sample.

Serial Dilution: Formula, Calculator, Method

Aim& Objective
 The objective of the serial dilution method is to estimate the concentration (number of
organisms, bacteria, viruses, or colonies) of an unknown sample by the enumeration of the
number of colonies cultured from serial dilutions of the sample.
 In serial dilution, the density of cells is reduced in each step so that it is easier to
calculate the concentration of the cells in the original solution by calculating the total dilution
over the entire series.
 Serial dilutions are commonly performed to avoid having to pipette very small
volumes (1-10 µl) to make a dilution of a solution.
 By diluting a sample in a controlled way, it is possible to obtain incubated culture
plates with an easily countable number of colonies (around 30–100) and calculate the number
of microbes present in the sample.

Formula and calculation

 Serial dilution involves the process of taking a sample and diluting it through a series of
standard volumes of sterile diluent, which can either be distilled water or 0.9 % saline.
 Then, a small measured volume of each dilution is used to make a series
of pour or spread plates.
 Depending on the estimated concentration of cells/organisms in a sample, the extent of
dilution is determined. For e.g., if a water sample is taken from an extremely polluted
environment, the dilution factor is increased. In contrast, for a less contaminated sample, a
low dilution factor might be sufficient.
 Serial two-fold and ten-fold dilutions are commonly used to titer antibodies or prepare
diluted analytes in the laboratory.
 The dilution factor in a serial dilution can be determined either for an individual test
tube or can be calculated as a total dilution factor in the entire series.
 The dilution factor of each tube in a set:

For a ten-fold dilution, 1 ml of sample is added to 9 ml of diluent. In this case, the dilution
factor for that test tube will be:

 After the first tube, each tube is the dilution of the previous dilution tube.
Now, for the total dilution factor,
 Total dilution factor for the second tube = dilution of first tube × dilution of the
second tube.
Example:
For the first tube, dilution factor = 10-1 (1 ml added to 9 ml)
For the second tube, dilution factor = 10-1 (1ml added to 9 ml)
Total dilution factor = previous dilution × dilution of next tube
= total dilution of 10-1 × 10-1 = 10-2

Procedure
The following is the procedure for a ten-fold dilution of a sample to a dilution factor of 10 -6:
1. The sample/culture is taken in a test tube and six test tubes, each with 9 ml of sterile
diluents, which can either be distilled water or 0.9% saline, are taken.
2. A sterile pipette is taken.
3. 1 ml of properly mixed sample/culture is drawn into the pipette.
4. The sample is then added to the first tube to make the total volume of 10 ml. This provides
an initial dilution of 10-1.
5. The dilution is thoroughly mixed by emptying and filling the pipette several times.
6. The pipette tip is discarded, and a new pipette tip is attached to the pipette.
7. Now, 1 ml of mixture is taken from the 10 -1 dilution and emptied into the second tube. The
second tube now has a total dilution factor of 10-2.
8. The same process is then repeated for the remaining tube, taking 1 ml from the previous
tube and adding it to the next 9 ml diluents.
9. As six tubes are used, the final dilution for the bacteria/cells will be 10-6 (1 in 1,000,000).
Application of serial dilution
Serial dilution is performed in several experimental sciences like biochemistry,
pharmacology, physics, and homeopathy.
1. Serial dilution is used in microbiology to estimate the concentration or number of
cells/organisms in a sample to obtain an incubated plate with an easily countable number of
colonies.
2. In biochemistry, serial dilution is used to obtain the desired concentration of reagents and
chemicals from a higher concentration.
3. In pharmaceutical laboratories, serial dilution is performed to receive the necessary
concentration of chemicals and compounds, as this method is more effective than individual
dilutions.
4. In homeopathy, homeopathic dilutions are used where a substance is diluted in distilled
water or alcohol. It is believed that dilution increases the potency of the diluted substance by
activating its vital energy.
Limitation of serial dilution
Even though serial dilution is a useful technique in laboratories, it faces some challenges.
Some of these are:
1. An error might occur during the propagation of the sample, and the transfer inaccuracies
lead to less accurate and less precise transfer. This results in the highest dilution to have the
most inaccuracies and the least accuracy.
2. Because serial dilution is performed in a stepwise manner, it requires a more extended
period which limits the efficiency of the method.
3. Serial dilution only allows the reduction of bacteria/cells but not the separation of
bacteria/cells like in other techniques like flow cytometry.
4. This technique also requires highly trained microbiologists and experts in aseptic
techniques.
Reference
 Basic Practical Microbiology-Manual. The Society of General Microbiology. Retrieved
from https://microbiologyonline.org/file/7926d7789d8a2f7b2075109f68c3175e.pdf
 Avishai Ben-David, Charles E. Davidson, Estimation method for serial dilution
experiments, Journal of Microbiological Methods, Volume 107, 2014, Pages 214-221, ISSN
0167-7012,
 Cullen, J. J., & MacIntyre, H. L. (2016). On the use of the serial dilution culture method to
enumerate viable phytoplankton in natural communities of plankton subjected to ballast water
treatment. Journal of applied phycology, 28(1), 279–298. https://doi.org/10.1007/s10811-
015-0601-x
 https://www.biomol.com/dateien/Bethyl–Serial-Dilutions.pdf
 https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/
Microbiology_Labs/Microbiology_Labs_I/04%3A_Dilution_Worksheet_and_Problems

Colony Morphology
Since bacteria were first cultured on solid media, describing the appearance of bacterial
colonies has been an important tool for microbiologists. Observing colony morphology is a
tool used by clinical microbiologists, in particular, and descriptions of colonies are often
found in the primary literature. Distinguishing colony morphology is one of the first skills
taught to microbiology students.
The colony morphology of bacteria on nutrient agar can vary depending on the type of
bacteria and can include characteristics such as:
 Shape: Colonies can be circular, convex, or other shapes
 Size: Colonies can be large or small, with a diameter of 2–4 mm
 Color: Colonies can be white, grayish, golden yellow, or fuzzy white
 Surface: Colonies can be smooth, rough, dull, glistening, or wrinkled
 Elevation: Colonies can be observed from a side angle to note their elevation

Procedure
Cultures of microorganisms can be grown on any medium that is appropriate for their
isolation and cultivation. Since morphology is influenced by medium type and growth
conditions, care should be taken to record these parameters. Good determination of colony
morphology is predicated on good streak technique because it requires good separation of
colonies. Smibert and Krieg (5) proposed the following protocol: 1. Measure the colony
diameter in millimeters. 2. Describe the pigmentation (distinguishing between pigmented
colonies and those secreting diffusible pigments). 3. Describe the form, elevation, and margin
as indicated in Fig. Also indicate whether the colonies are smooth (shiny glistening surface),
rough (dull, bumpy, granular, or matte surface), or mucoid (slimy or gummy appearance). 4.
Record the opacity of the colonies (transparent, translucent, or opaque) and their texture
when tested with a needle: butyrous (buttery texture), viscous (gummy), or dry (brittle or
powdery).