Microbes in Water - Determination of the Cell Number

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Sophomore (College 2nd year) ・Art ・MLA ・16 Sources

Aim: An experiment to determine the number of microbes in a water sample.
Procedure
Materials needed
Cultures: Water sample
Media: nutrient agar plates, ENDO agar plates
Buffer: 0.75% (w/v) sodium chloride
Reagents: sterile water
Apparatus needed
Membrane filter gear including filter funnel (100-500 ml)
Filter support
Filter base
Receiver flask
Vacuum source
Sterile membrane filter (Pore wideness: 0.2 μm)
Forceps
Sterile pipettes
Tubes
Drigalski spatula
Experiment Theory

Sample Drawing

The sample of water was collected in a sterile vessel that was retrieved from the lab on the first day of the practical course. The water sample was collected from a small creek on 3rd May 2017 at 23:37 pm. The creek is located in Hurdel region of Bonn-Duisdorf, in a surrounding with human beings, dogs, cats, and birds.

Determination of the Viable Count, Using the Spread Plate Method

  1. A serial dilution was prepared using 4 ml of NaCl and 15 ml of water. 0.1 ml of each dilution and original sample were transferred onto the agar plates with different amounts of dilution.
  2. The Sample was distributed using the Drigalski spatula. A sterile spatula was used for different concentrations of the sample into the different plates.
  3. The plates were incubated aerobically at room temperature at 13: 30 hours.

Determination of the Coliform, Using the Membrane Filter Technique

  1. Each sample was filtered and dilutions 10­-2 and 10-1 were added.
  2. The filters were transferred without any air bubbles onto the ENDO agar plates with the grid facing the observer.
  3. The plates were incubated right side up at 37oC for 18-22 hours. They are incubated right side up in order to expose the sample to greater air flow.

Note: Every step was followed in accordance with the script. There were no modifications of materials used or any procedural methods.

After the Work

The spatula was disinfected after usage for 25 minutes, in the Helipur H plus N, separately.

The filtration equipment: The forceps, funnel, and filter support were stored in the biohazard bag and the bag left open. The filter base and receiving flask were stored separately from each other, with the former having the gasket. Filtered water was discarded in the sink and the equipment left to dry

            Evaluation

Results

            Determination of Viable Count: Spreading Plate Method

Determination of Viable Count: Spreading Plate Method

Organism Type

Color

Morphology (form; margin; elevation)

A

dark purple

Irregular; flat

B

red/orange

circular; clear; flat

C

yellow/grey

filamentous; lobate

D

yellow/grey

circular; entire; raised

E

yellowish grey

circular; entire; raised

F

grey

Irregular; lobate; flat

G

yellow

circular; curled; umbonate

H

yellow

circular; entire; flat

I

yellow

Rhizoid; spreading; raised

 

Cell Count

Organism Type

100

10-1

10-2

10-3

10-4

A

Uncountable;

 

 

 many;

 

 

clustered;

 

2

many

many

Uncountable;

 

 

 many;

 

 

clustered;

 

B

Uncountable;

 

 

 many;

 

 

clustered

many

1

C

2

1

D

many

~6

E

many

5

F

many

3

G

~50

2

H

~6

many

I

many

many

Determination of the Coliform, Using the Membrane Filter Technique

Organism Type

Color

Morphology (form; margin; elevation)

A

Orange

Circular; entire

B

Greenish

circular; entire; convex

C

Dark purple/red

filamentous; filamentous; convex

 

Cell Count

Organism Type

100

10-1

A

122

43

B

61

19

C

3

1

Discussion

Experiment Theory

The purity of water is determined by a number of factors. Microorganisms, chemicals, and particles are impurities and it is, thus, important to determine their percentages for various purposes (Beran 2010, 232). In some instances such as food processing and formation of fertilizers bacteria and other microorganisms play a greatly important role (Trigiano and Gray 2016, p. 157). The same case applies for research involving the development of cures for various diseases (Vishwakarma & Karp 2017, p. 602). These applications demand the constant availability of the microorganisms in plenty, which calls for their mass production (Chauhan 2009, p. 370).

The growth of microorganisms is an important component. Its measurement is essential as it helps to determine the number of organisms within a particular water body or any other niche (Pepper, et al. 2011, p. 174). There are numerous ways of determining the number of microbial organisms, which are divided into two: direct and indirect techniques (Gopal 2004, p. 123). In the former, the microbes are placed on a slide and counted physically by a number of methods such as the plate count, microscopic count, determination of the most probable number (MNP), and membrane filtration.

Direct microscopic count utilizes specialized slides referred to as the counting chambers (Harrigan 1998, p. 73). Since the microbes are too small to determine whether the specimen is fully alive or partially dead, it is impossible to determine whether an area under observation has any living microbes (Prakashan n.d, p. 4.20). Therefore, this type of count concentrates on dense suspensions within the specimen. Its sensitivity is usually taken as multiples of 10 million microbes per ml.  Filtration and centrifugation are the two techniques that may be used to increase the concentration of the sample, hence, the sensitivity of the count (Nair 2010, p. 290). There are various variations of this type of count, which have been devised over the years of research in order to fit particular environments or settings. For instance, the growth of thermophilic bacteria in hot springs demanded the immersion of the observation slides into the boiling springs and their periodical withdrawal for microscopic observation as studied by Dr. T. D Brocks (Nair 2010, p. 381). Once the bacteria form micro-colonies on the glass plate, it is taken out and a count conducted to obtain a value that is used to estimate their concentration (Lengeler, et al. 2009, p. 101).

Electronic counting chambers is a method of counting microorganisms by their size distribution and numbers using electronic equipment such as the electron microscope (Pal & Pal 2006, p. 37). It is usually utilized as a method for counting eukaryotic organisms or cells such as the blood cells. However, whenever it is used in bacterial count, then the suspension medium has to be clear (Heaney, et al. 1994, p. 1528).

Indirect viable cell count is also referred to as the plate count. This method involves the spreading of a culture sample on a surface with nutrient agar. Before plating, the sample suspension may be diluted using a non-toxic diluent such as saline or water (Elsevier 1988, p. 9). The cells form colonies form each viable unit if a suitable medium is used. The separate colonies that are distinguishable are referred to as colony forming units (CFU). Each unit represents the number of viable clusters of bacteria within the sample (Greer 2009, p. 107).

This lab process is fundamental in science because it serves both public and government interests. Water that is to be used for any applications such as drinking and domestic or industrial purposes has to be tested to determine how safe it is for usage (Eslamian 2016, p. 515). The standard procedures have to be followed for the sake of reproducibility for confirmation. However, every circumstance calls for particular modifications that have to be documented for the same purpose of reproducing the results.

Determination of Viable Count: Spreading Plate Method

There were nine types of microorganisms identified into 5 different colonies that were grown. Organism identified as G dominated the plate with an approximate count of 50. The sample contained an uncountable number of microorganisms, most of which could hardly be identified specifically. The presence of an innumerable number of organisms lowers the quality of water. Therefore, the water is unsafe for consumption.

Determination of the Coliform, Using the Membrane Filter Technique

Only two colonies were considered for three types of organisms. From the experiments above, Coliform bacteria and E. coli have been determined to be present owing to the colors of the specimen. The two organisms metabolize lactose to produce an acid and aldehyde with the latter producing fuchsin from compounds of fuchsin-sulfite. Fuchsin is responsible for the color red in the colonies. E. coli colonies cause the crystallization of this chemical thus producing a greenish fuchsin sheen. Therefore, Organism B is identified as E. coli while C is the coliform bacteria. Organism A is unidentified.

The two microorganisms, E. coli and coliform were identified based on the reference to class notes offered from the theoretical perspective. The colors of the sample media changed as expected, thus enabling easy identification of the organisms. Organism A was not identified as there was no reference of its characteristics in class readings.

References

Beran, J. A., 2010. Laboratory Manual for Principles of General Chemistry. s.l.:John Wiley & Sons.

Chauhan, A. K., 2009. A Textbook of Molecular Biotechnology. s.l.:I. K. International Pvt Ltd.

Elsevier, 1988. Chapter 2: Methods of cell counting and assaying cell viability. Laboratory Techniques in Biochemistry and Molecular Biology, Volume 18, pp. 7-17.

Eslamian, S., 2016. Urban Water Reuse Handbook. s.l.:CRC Press.

Gopal, K., 2004. Fundamentals of Water and Waste Water. s.l.:APH Publishing.

Greer, J. P., 2009. Wintrobe's Clinical Hematology, Volume 1. s.l.:Lippincott Williams & Wilkins.

Harrigan, W. F., 1998. Laboratory Methods in Food Microbiology. s.l.:Gulf Professional Publishing.

Heaney, L., McKirgan, J., Stanford, C. & Ennis, M., 1994. Electronic cell counting to measure total cell numbers in bronchoalveolar lavage fluid.. Eur Respir J., 7(8), pp. 1527-31.

Lengeler, J. W., Drews, G. & Schlegel, H. G., 2009. Biology of the Prokaryotes. s.l.:John Wiley & Sons.

Nair, A., 2010. Principles of Biochemistry and Genetic Engineering. s.l.:Laxmi Publications.

Nair, A. J., 2010. Comprehensive Biotechnology XI. s.l.:Firewall Media.

Pal, G. & Pal, P., 2006. Textbook Of Practical Physiology - 2Nd Edn.. s.l.:Orient Blackswan.

Pepper, I. L., Gerba, C. P., Gentry, T. J. & Maier, R. M., 2011. Environmental Microbiology. s.l.:Academic Press.

Prakashan, N., n.d. basic microbiology for nursing and health science. [Online]
Available at: https://books.google.co.in/books?id=_Xw39Hb65lMC&pg=SA4-PA19&dq=Direct++microscopic+count&hl=en&sa=X&ved=0ahUKEwiTvLezs-vTAhVFPY8KHTPlBLcQ6AEIQTAG#v=onepage&q=Direct%20%20microscopic%20count&f=false
[Accessed 13 May 2017].

Trigiano, R. N. & Gray, D. J., 2016. Plant Tissue Culture, Development, and Biotechnology. s.l.:CRC Press.

Vishwakarma, A. & Karp, J. M., 2017. Biology and Engineering of Stem Cell Niches. s.l.:Academic Press.

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