Bacteria Enumeration - Definition, Methods & Example - Conduct Science (2023)

What is Bacteria Enumeration?

Enumeration of bacteria is defined as the process of determining the number of bacteria in a given sample.

There are numerous reasons why researchers have to calculate the number of bacteria or compare the growing number of these microbes under certain specific conditions. It’s mainly essential and a part of routine work in food, water, and dairy microbiology labs.[1]

The knowledge of the numbers of these microorganisms in our food, including milk, curd, buttermilk, and water, help the labs to determine if the prepared food or available water is hygienic or not for consumption.

However, as we know, these microorganisms are so small in size and are invisible to our naked eyes; thus, a high-power microscope is required to study these organisms. Hence, counting them is often a pain in the neck.

But thankfully, scientists have developed high-throughput protocols to make it easier to count the numbers of these organisms in any given sample.

This article discusses the techniques used to count bacteria in food or water samples and the advantages and disadvantages of these microbiological techniques.

Categories of Bacteria Enumeration

Bacteria enumeration is divided into four categories:[2]

1. Direct Cell Count:

This process counts the number of bacteria in a liquid media or colonies on a plate. In this process, counting chambers is used to directly count the bacterial numbers under the microscope.

2. Indirect Cell Count:

It’s the process of estimation of bacterial numbers. This process doesn’t involve direct counting of bacterial cell numbers, however, bacterial colonies are calculated through which the concentration of bacterial cells is estimated in the given sample. It’s done by the plate count method.

(Video) LECTURE 26: ENUMERATION OF MICROORGANISMS

3. Viable Cell Count:

It’s counting bacterial cells that are metabolically active and actively dividing and growing in the culture medium.

Involves counting the total bacterial cell numbers, including both metabolically active and metabolically inactive/dead microbes.[2]

These four categories of estimating bacteria population are combined in four ways based on the purpose of experimentation in labs. It includes:[2]

  • Direct/Viable: It’s a standard plate count method in which repeated dilutions of a sample determine the number of viable bacteria cells in the given sample. It’s used while calculating the number of bacteria in cellular vaccines or milk.
  • Indirect/Viable: This is a statistical inference about the microbial count based on growth patterns. It’s taken into consideration while enumerating the bacterial concentration in soil, laboratory cultures, or water.
  • Direct/Total: Here, dyes and fluorescent stains are used to make the bacterial cells visible using a fluorescent microscope to aid in counting the bacteria population in the given sample.[2] It’s utilized while counting the numbers of bacteria in aquatic samples.
  • Indirect/Total: Spectroscopy is considered as indirect/total enumeration. With a spectrophotometer, the amount of light passed through the culture is used to estimate the number of microbes present.[2] It’s used to estimate the concentration of bacteria in food samples.

Techniques for Bacteria Enumeration

The determination of bacterial cell numbers can be achieved by many methods, including culture turbidity, dry weight of cells, and direct microscopic count. However, among these, the direct viable count technique is the one that’s commonly used.[3]

Below are some frequently used bacteria enumeration techniques, their principles, and their differences.

1.Standard Plate Count (Viable Counts)

Bacteria can be present in thousands or millions in a sample, making it difficult to count their numbers. However, serially diluting the cultures makes it easier to determine the count.

After serial dilution, the aliquots of the diluted sample are plated on an appropriate culture media. Then, the plates are incubated, after which the number of colonies formed is counted. This technique is also known as plate count or colony counts.[3]

Bacteria Enumeration - Definition, Methods & Example - Conduct Science (1)
(Video) Viable cell count

The techniques that assist in standard plate count include the streak plate and pour plate methods. It’s essential to ensure that the plate is not crowded with bacterial colonies because, in such conditions, some cells might either not form colonies or fuse with each other resulting in erroneous results. Statistically, it is most valid to count colonies only on plates with 30 to 300 colonies.[3]

In this method, accurate determination of total cell numbers is only possible if each colony is formed of a single cell. However, it’s difficult for one to ensure such a case; that’s why the total numbers of cells reported using this method are termed Colony Forming Units (CFUs) rather than cell numbers. It’s calculated as:[3]

The number of CFUs per ml of sample = The number of colonies (30-300 plate) X The dilution factor of the plate counted.

Advantages of plate count method

  • Feasible for enumerating bacteria in milk, water, foods, soils, cultures, etc.
  • Relatively simple to use and more sensitive than turbidimetric measurement.
  • It allows positive identification of the organism counted.
  • It’s easy to count small numbers of bacteria in a sample using this method.

Limitations of plate count technique

  • It’s a time-consuming process where every stage, including dilutions, platings, incubations, and media preparation, requires a fixed amount of time.[3]
  • Only living cells develop colonies; therefore, this method can only count them.
  • Clumps or chains of cells can develop into a single colony, leading to erroneous results.
  • A colony can only arise from organisms that can grow under the provided conditions. This can lead to miscalculation.

2.Turbidimetric Measurement

Turbidity measures the loss of intensity of transmitted light due to the scattering effect of suspended particles.[5] It’s simply the amount of cloudiness or turbidity in the sample. It can be caused by silt, sand, mud bacteria, and other microbes or chemical precipitates.

The turbidimetric method is a quick and efficient method to measure and estimate the number of bacteria in a given sample. The method is most preferable when large numbers of cultures are required to be counted.

When bacteria are mixed in a liquid medium, it creates a colloidal suspension that blocks and reflects light as it passes through the culture. The light absorbed by bacterial suspension will estimate the concentration of bacteria in the given sample.

You must note that “the light absorbed by the culture is directly proportional to the cells’ concentration.[5]

Even though the turbidity measurement technique is faster than the standard plate count, it is initially required to match the measurement values with cell numbers, which requires lab personnel to combine the standard plate count method with the turbidity technique. The procedure followed include:[5]

  • First, the turbidity of different concentrations of a given bacterial species in a culture medium is determined.
  • Using standard plate count, the number of viable microorganisms per milliliter of the samples is estimated.
  • A standard curve is drawn, with turbidity or optical density readings corresponding to specific numbers of living organisms.

Once drawn, the standard graph can be directly used for subsequent turbidity measurement and calculating the number of viable bacterial cells, eliminating the process of time-consuming standard plate count.

The method utilize instruments such as a spectrophotometer or colorimeter to measure the turbidity in the given sample. They contain a light source and a light detector separated from the sample compartment.[3]

The cultures are poured into a cuvette and kept in the instrument. When light passes through the culture sample, cells interfere with them, which provides an output value on the machine that helps to calculate the bacteria numbers in the cultures.

Bacteria Enumeration - Definition, Methods & Example - Conduct Science (2)

Before experimenting, it’s essential to adjust the spectrophotometer at 100% transmittance (which means 0% absorbance), which is done using a sample of the uninoculated medium.

At last, the percentage of transmittance of different bacterial culture dilutions is measured, and the obtained values are converted to optical density using the formula:[1]

Absorbance (O.D.) = 2 – log % Transmittance

The wavelength is determined based on the color of the solution. For example, if it’s white, 420 nm is used, 540 nm if the color is yellow[1], and 600-625 nm if the solution is in the range of yellow to brown color.

(Video) Outline of Enumeration of Bacteria

Advantages of turbidimetric measurement

  • It’s a faster procedure than the standard plate count technique.
  • It can be done without spoiling the samples.

Limitations of turbidimetric measurement

  • For the estimation, it requires a high amount of bacterial cells—about 100 million cells per milliliter.
  • If the mass of bacteria increases more than normal in the sample, they mask the light passed through the cuvette, resulting in inaccurate calculations.

3.Direct Microscopic Count

The direct microscopic count is used for quantitative enumeration of bacteria in water, food, milk, and air samples.[3]

It is done by spreading a measured volume of the sample over a predetermined area of a slide, counting representative microscopic fields, and transforming the average values into appropriate volume-area factors.[3]

The counting chambers used for the procedures include Petroff-Hauser and Levy counting chambers.[3]

The Petroff-Hauser is a thick microscope slide with a chamber in the middle of 0.02 mm (1/50 mm) deep, and it also has improved Neubauer rulings and an etched grid in the chamber. The ruling is the centerline of a group of three squares in which cells are counted.

Bacteria Enumeration - Definition, Methods & Example - Conduct Science (3)

A single drop of culture is applied in this counting method using a Pasteur pipette. The cell numbers in the given sample are counted directly in 10-20 high microscope fields. Based on the average number of cells per field, the number of bacteria per milliliter of the original sample can be obtained.

The final concentration of cells in the given sample is calculated using the formula:[3]

(Video) lab#5: Enumeration of microbial cells

Total cells counted x 2.0×10 7 x dilution factor/ # small squares counted = cells/ml

Using fluorescent dyes

The direct microscopic count is also done using fluorescent dyes. These dyes are popular in labs because of their ability to stain all bacterial species in a given sample, a particular species, or a specific component of cells. A few examples of fluorescent dyes are cyanoditolyl tetrazolium chloride (CTC), auramine, acridine orange, and rhodamine.

The most common fluorescent dye used to stain bacteria is acridine orange. In this method, a known sample volume is stained with acridine orange and then filtered through a 0.22 µm filter. The filter traps the bacteria that are then examined under the fluorescent microscope. By counting bacteria in a defined area of the filter, the concentration of bacteria in the original sample can be determined.

Advantages of of direct microscopic count

  • Direct counting procedures are rapid, simple, and easy.
  • You can observe the morphology of the bacteria while counting the numbers.
  • After dilutions, even dense suspensions can also be counted using the technique.

Limitations of direct microscopic count

  • They do not discriminate between living and dead cells.
  • It can not be used to count viable cells.
  • You can’t observe small cells, so they can be missed during counting.
  • This method cannot be used with cell suspensions of low density, i.e., <107 cells per ml.

Conclusion

Bacteria enumeration is the process of determining the number of bacterial cells in a given sample. The counting of bacterial cells has four categories based on the purpose of the experiment: direct, indirect, viable, and total cell count. The categories are combined in four different ways to serve the experimental purpose in different techniques.

Many methods have been developed to count the numbers of bacteria in labs, but the most frequently used ones are standard plate count, turbidimetric method, and direct microscopic count.

The techniques are essential in food and beverage industries, where counting the numbers of bacteria in the given food or beverage sample is essential to learn if they are safe to consume and are not contaminated. Besides this, bacteria enumeration also has applications in agriculture and processing industries.

Despite making the enumeration process easier and smoother, the available techniques have several limitations. Thus offering young researchers the chance to develop techniques that cover the limitations and provide a faster approach for the enumeration process.

References:

  1. Bacteria enumeration. Retrieved from https://www.slideshare.net/martyynyyte/bacteria-enumeration-37491510.
  2. Libel angela. What Is Enumeration in Microbiology? Retrieved from https://sciencing.com/what-is-enumeration-in-microbiology-12760382.html
  3. Tankeshwar Acharya. Isolation and Enumeration of Bacteria. Retrieved from https://microbeonline.com/techniques-of-isolation-and-enumeration-of-bacteria/.
  4. Tankeshwar Acharya. Pour Plate Method: Procedure, Uses, (Dis) Advantages. Retrieved from https://microbeonline.com/pour-plate-method-principle-procedure-uses-dis-advantages/
  5. Bacterial Enumeration: Definition, Methods & Example. Retrieved from https://study.com/academy/lesson/bacterial-enumeration-definition-methods-example.html#

FAQs

What are the methods of enumeration of bacteria? ›

The most common procedure for the enumeration of bacteria is the viable plate count. In this method, serial dilutions of a sample containing viable microorganisms are plated onto a suitable growth medium.

What are 3 methods used to identify bacteria? ›

Modern Methods for Identifying Microbes
  • Identifying Microbes Using PCR. PCR, including Real-Time PCR, is probably the most widely used molecular technique for identifying microbes. ...
  • Microarray-Based Identification. ...
  • Immunological Identification. ...
  • Chemical/Analytical Identification.
27 Jul 2017

What is the method of bacteria? ›

Methods of Bacterial Culture

Agar Plates: Scientists can grow the bacterial culture in a petri dish containing a thin layer of agar medium. The agar plates are inoculated with the desired bacteria strain and stored in an incubator overnight at an optimal temperature that is suited for the bacteria's growth.

Which method is used for enumeration of bacteria in vaccines and cultures? ›

THE NEPHELOMETER:AN INSTRUMENT FOR ESTIMATING THE NUMBER OF BACTERIA IN SUSPENSIONS USED FOR CALCULATING THE OPSONIC INDEX AND FOR VACCINES.

What is the methods of enumeration? ›

Bacteria enumeration is the process of determining the number of bacterial cells in a given sample. The counting of bacterial cells has four categories based on the purpose of the experiment: direct, indirect, viable, and total cell count.

What is an enumeration example? ›

Enumeration means counting or reciting numbers or a numbered list. A waiter's lengthy enumeration of all the available salad dressings might seem a little hostile if he begins with a deep sigh. When you're reciting a list of things, it's enumeration.

What are 4 types of bacteria? ›

There are four common forms of bacteria-coccus,bacillus,spirillum and vibrio.

What are two methods used to identify a species of bacteria? ›

Bacteria are identified routinely by morphological and biochemical tests, supplemented as needed by specialized tests such as serotyping and antibiotic inhibition patterns. Newer molecular techniques permit species to be identified by their genetic sequences, sometimes directly from the clinical specimen.

What is the most common method of identifying bacteria? ›

DNA sequencing is the gold standard for microorganism identification. The 16S ribosomal RNA (rRNA) gene is the most common sequencing target for bacteria and is 1542 base pairs (bp) in length.

Which method is used for growth of bacteria? ›

Bacterial growth is proliferation of bacterium into two daughter cells, in a process called binary fission. Providing no event occurs, the resulting daughter cells are genetically identical to the original cell. Hence, bacterial growth occurs.

What are two methods of measuring bacterial growth? ›

The two most common classroom methods to determine bacterial growth are the Standard Plate Count (SPC) technique and turbidimetric measurement. Examples of other methods include: microscopic count, membrane filter count, nitrogen determination, cellular weight determination, and biochemical activity measurement.

What is bacteria method of reproduction? ›

Most bacteria rely on binary fission for propagation. Conceptually this is a simple process; a cell just needs to grow to twice its starting size and then split in two.

What are the methods for enumeration of microbes in food? ›

Five methods for the enumeration of micro-organisms in food (pour plate, surface spread plate, surface drop, agar droplet, and microdilution) were used in parallel to examine 100 samples selected from a wide range of food products.

What are the three methods of inoculating bacteria on agar? ›

Different methods of inoculation include stab culture, slant culture, agar culture and broth culture.

Which device is used for the enumeration of bacteria in air? ›

Explanation: Anderson' air sampler is a devise that is used for the enumeration of bacteria in air. This device is used in microbiology for bacterial enumeration to count the no of bacteria. One other device is also used in somethimes that is capillary impinge.

How many methods of enumeration are there? ›

It has four constants SMALL , MEDIUM , LARGE and EXTRALARGE . Since Size is an enum class, the compiler automatically creates instances for each enum constants. Here inside the main() method, we have used the instance SMALL to call the getSize() method.

What is enumeration and its types? ›

Enumeration is defined as the process of extracting user names, machine names, network resources, shares and services from a system. In this phase, the attacker creates an active connection to the system and performs directed queries to gain more information about the target.

What are the types of enumerate? ›

The most prevalent forms of enumeration include:
  • NetBIOS Enumeration. ...
  • SNMP Enumeration. ...
  • LDAP Enumeration. ...
  • NTP Enumeration. ...
  • SMTP Enumeration. ...
  • DNS Enumeration. ...
  • User Enumeration using Email IDs and Usernames. ...
  • Enumeration Using Default Passwords.
9 May 2022

What are the two types of enumeration? ›

enumeration types, including the predefined types boolean, bit and character.

What is used to list examples and enumeration? ›

The keyword 'enum' is used to declare new enumeration types in C and C++. Following is an example of enum declaration.

What is a meaning of enumeration? ›

: the act or process of making or stating a list of things one after another. the rebel leader's effective enumeration of popular grievances. also : the list itself.

What are 5 examples of bacteria? ›

Examples include Listeria monocytogenes, Pesudomonas maltophilia, Thiobacillus novellus, Staphylococcus aureus, Streptococcus pyrogenes, Streptococcus pneumoniae, Escherichia coli, and Clostridium kluyveri.

What are the 10 examples of bacteria? ›

Top Ten Bacteria
  • Deinococcus radiodurans.
  • Myxococcus xanthus. ...
  • Yersinia pestis. ...
  • Escherichia coli. ...
  • Salmonella typhimurium. ...
  • Epulopiscium spp. The big boy of the kingdom – about as large as this full stop. ...
  • Pseudomonas syringae. Dreaming of a white Christmas? ...
  • Carsonella ruddii. Possessor of the smallest bacterial genome known, C. ...
5 Dec 2008

What are examples of bacteria? ›

Bacteria

What are the methods used to identify species? ›

Methods used for the identification of species are mainly based on protein and DNA analysis, with protein characterization being carried out using immunological, electrophoretical, and chromatographic techniques (Mafra et al., 2008).

What are the direct methods used for determining bacterial counts? ›

Direct counting methods include microscopic counts using a hemocytometer or a counting chamber. The hemocytometer works by creating a volumetric grid divided into differently sized cubes for accurately counting the number of particles in a cube and calculating the concentration of the entire sample.

What tests are used to identify bacteria? ›

Techniques like straining of isolated bacteria, motility testing, Biochemical testing, Serological tests, Phage typing, identification disc testing, Semiautomated and Automated identification system & Molecular techniques are used for bacterial identification. also carry out antibiotic susceptibility testing.

What 4 characteristics are used to identify bacteria? ›

Indeed, bacteria are classified on the basis of many characteristics. Cell shape, nature of multicell aggregates, motility, formation of spores, and reaction to the Gram stain are important.

What are characteristics of bacteria? ›

There are three notable common traits of bacteria, 1) lack of membrane-bound organelles, 2) unicellular and 3) small (usually microscopic) size. Not all prokaryotes are bacteria, some are archaea, which although they share common physicals features to bacteria, are ancestrally different from bacteria.

What are the properties of bacteria? ›

Five characteristics of bacteria include being unicellular, prokaryotic, microscopic, lacking a nucleus, and having a plasma membrane. These traits are shared by all bacteria.

What are the 4 stages of bacterial growth? ›

It has been determined that in a closed system or batch culture (no food added, no wastes removed) bacteria will grow in a predictable pattern, resulting in a growth curve composed of four distinct phases of growth: the lag phase, the exponential or log phase, the stationary phase, and the death or decline phase.

What are the four process of bacteria growth? ›

The bacterial growth progresses in four phases namely – lag phase, log phase, stationary phase and death phase.

What are the types of culture methods? ›

  • Streak culture or surface plating.
  • Lawn or carpet culture.
  • Stroke culture.
  • Stab culture.
  • Pour plate method.
  • Anaerobic methods of culturing bacteria.

What is used to measure bacteria? ›

If you are referring to what unit is used in measuring bacteria the answer would most commonly be micrometers (µm).

Which instrument is used to measure bacteria? ›

The spectrophotometer measures the turbidity or optical density. Turbidity is the measure of the amount of light absorbed by a bacterial suspension. The degree of turbidity in the broth culture is directly proportional to the number of microorganism present i.e. microbial density.

Which method is used for measurement of growth? ›

Cell count this method involves the measurement of growth either by microscopy or by using an electronic particle counter or indirectly by a colony count.

Which is an example of bacteria reproduction? ›

Binary Fission – It is a common and simple method of asexual reproduction in bacteria. In this process of asexual reproduction, the parent cell divides into two, leading to two equal-sized daughter cells.

What are the 10 methods of reproduction? ›

The organisms reproduce in two ways: Asexual Reproduction– In this process, only a single parent is involved and no gamete formation takes place. Sexual Reproduction– In this process, two parents are involved and gamete formation takes place. Meiosis is an important step in sexual reproduction.

What are the five methods of reproduction? ›

The different methods of asexual reproduction are:
  • Fission.
  • Budding.
  • Spore formation.
  • Regeneration.
  • Fragmentation, and.
  • Vegetative reproduction.

What are 4 methods of food getting in bacteria? ›

Bacteria can obtain energy and nutrients by performing photosynthesis, decomposing dead organisms and wastes, or breaking down chemical compounds. Bacteria can obtain energy and nutrients by establishing close relationships with other organisms, including mutualistic and parasitic relationships.

Which method is used for the enumeration of coliform bacteria? ›

Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water. The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these methods.

What is an example of inoculation? ›

Inoculation describes the process of deliberately infecting an unexposed person with a mild strain (for example variola minor) of smallpox to create a mild form of the disease. Post inoculation, the individual was left with immunity against smallpox.

What are the types of inoculation? ›

Types of Inoculation
  • Direct Inoculation.
  • Covert Inoculation.
  • Indirect Inoculation.
  • Formal Inoculation.
  • Informal Inoculation.
  • Subliminal Inoculation.

What are the 3 types of bacterial culture media? ›

Based on consistency culture media is classified as liquid, semi-solid and solid media.

What is the purpose of microbial enumeration test? ›

This test is to determine mesophilic bacteria and fungi that grow under aerobic conditions. Different incubation temperature and media are required for the growth of bacteria and fungi.

What is rapid method of enumeration of microorganisms? ›

Ganatra: Rapid microbial methods (RMMs), also known as alternative microbiological methods, are the technologies that allow the user to get microbiology test results faster compared with traditional culture-plate methods. Miller: "Faster" means in a matter of hours, as opposed to days or weeks in some cases.

Which type of enumeration method uses an agar plate? ›

Whilst the pour plate method using plate count agar is regarded as the standard international method of enumeration for a total aerobic colony count, it is common for laboratories to use surface methods such as the surface drop and spiral plate.

What are 3 ways to measure bacterial growth? ›

The two most common classroom methods to determine bacterial growth are the Standard Plate Count (SPC) technique and turbidimetric measurement. Examples of other methods include: microscopic count, membrane filter count, nitrogen determination, cellular weight determination, and biochemical activity measurement.

What test is used to detect bacteria? ›

A bacteria culture test can help find harmful bacteria in or on your body that may be making you sick. To do the test, you will need to give a sample of your blood, urine, skin, or other tissue. The type of sample depends on where the infection seems to be located.

Which method is used for measuring bacterial growth? ›

The simplest way to count bacteria is called the direct microscopic cell count, which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope.

How do you collect bacteria samples? ›

Sampling Protocols
  1. Use sterile swabs. ...
  2. Remove the outer packaging of the sterile swab.
  3. It is often recommended to follow the instructions on the outer packaging to moisten the swab before sampling to help improve collection efficiency. ...
  4. Swab the area of concern. ...
  5. Return the swab into the tube.

Why is it important to identify bacteria? ›

Accurate and definitive microorganism identification, including bacterial identification and pathogen detection, is essential for correct disease diagnosis, treatment of infection and trace-back of disease outbreaks associated with microbial infections.

How do you identify bacteria under a microscope? ›

Another useful way of identifying a bacteria is by determining whether it is a gram negative or gram positive. This is achieved through the staining process and stains such as crystal violet dye, iodine, and the counterstain safranin.

Which bacteria is used in PCR? ›

PCR method. The major outer membrane protein (MOMP) genes, such as omp-1 (ompA) (32), of C. pneumoniae have been utilized widely in PCR as a target gene for detection of this bacterium.

What are the 4 types of infections? ›

Infections are common. From ear infections and the flu to COVID-19, chances are we all have had at least one at some point. Viral, bacterial, fungal, and parasitic infections can all trigger sepsis.

How do bacteria grow? ›

Bacteria grow and reproduces via binary fission, which involves the process of the parent organism growing and dividing into doubles into a new individual. The parent cell also shares identical DNA with the daughter cell after the elongation process. They consume nutrition and develop to reproduce.

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