Which Of The Following Can Be Used To Estimate The Number Of Microorganisms In A Culture


Counting Bacteria

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Learning Objectives

Methods of directly counting microorganisms that differ from one another

Key Takeaways

  • A hemocytometer, which counts blood cells or tissue cells in real time, may be used to calculate the concentration of a known amount of fluid. It is possible to compute the concentration of a solution by counting the number of colonies that appear on a pour plate and multiplying that count by a volume dispersed over the pour plate
  • However, this method is time-consuming. Direct counting methods are simple to use and do not necessitate the use of highly specialized equipment
  • Yet, they are significantly slower than other approaches.

Key Terms

  • Microparticle counter (hemocytometer): A device that counts tiny particles in blood. This device works by producing a volumetric grid divided into cubes of varying sizes, which can then be used to count the number of particles in each cube and calculate the overall concentration of a sample. In biology, a streak plate is a petri dish containing a growth media.

Direct Counting of Bacteria

Numerous operations in biology and medicine need the counting of cells in order to complete the task. Almost always, the concentration of cells is what is measured, rather than the total number of cells (for example: 5,000 cells per milliliter). The concentration of a culture may be determined by counting the number of cells in a known volume of the culture. As a result, in medicine, the concentration of various blood cells, such as those of red blood cells or white blood cells, can provide important information about a person’s health.

  1. In molecular biology research, knowing the cell concentration is critical since it allows you to control the amount of reagents and chemicals you use to complete the experiment correctly.
  2. Microscopical counts performed with a hemocytometer or a counting chamber are examples of direct counting procedures.
  3. The number of cells in a culture can also be determined by plating a known volume of the cell culture on a petri dish filled with growth media, which is known as a streak plate.
  4. The colonies may then be counted, and the cell concentration can be determined using the known volume of the culture that was put on the plate and the known number of colonies.
  5. Cultures must be highly diluted prior to plating, just as they must be with hemocytometers or counting chambers.
  6. Furthermore, plating is the slowest approach since most bacteria require at least 12 hours to develop visible colonies before they can be harvested.

Because these methods of direct counting do not necessitate the use of specialized gear, they may be conducted with relative ease in the majority of laboratories.

Viable Cell Counting

Plate counting is a technique that is used to estimate the number of live cells present in a given sample.

Learning Objectives

Explain the concept of viable cell counts.

Key Takeaways

  • With the spread plate, bacteria colonize an inert medium and develop to the point where the colony can be seen with the naked eye. This allows the number of colonies on a plate to be tallied. Selective media can be used to limit the development of germs that are not intended for usage. If you’re seeking for bacterial species that grow poorly in air, such as those found in water samples, the pour plate approach is what you’re looking for.

Key Terms

  • Plate count: A method of determining the number of cells that are actively proliferating in a sample

Viable Cell Counting

To prevent the development of non-target bacteria, selective medium can be utilized. The following image depicts a urine culture grown on Oxoid Brilliance UTI Agar plate. 1uL of urine was dispersed across the surface of the agar. The top sample comes from a patient who has been diagnosed with clinical urinary tract infection (UTI). The bottom sample contains a mixture of bacteria. There are several methods for counting the number of bacteria present in a sample. A viable cell count is a method of determining the number of cells that are actively growing and dividing in a sample.

  1. The colony is now visible to the human eye, and the number of colonies on a plate may be tallied using a simple counting method.
  2. When there are fewer than 30 colonies, the interpretation is statistically unsound, and when there are more than 300 colonies, there are often overlapping colonies and imprecision in the count.
  3. A typical laboratory approach entails diluting the sample in sterile water in successive dilutions (1:10, 1:100, 1:1000, etc.) and growing the resulting cultures on nutrient agar in a plate that is sealed and incubated for several days.
  4. coli and Salmonella.
  5. Typically, one set of plates is incubated at 22°C for 24 hours, while a second set of plates is incubated at 37°C for the same period.
  6. Some modern approaches make use of a fluorescent agent, which allows for the counting of colonies to be done automatically.
  7. The pour plate method is used when looking for bacterial species that do not grow well in air.

To begin, successive dilutions of the material are mixed in liquid nutrient agar, which is then put into bottles for further analysis.

After incubation, the number of colonies that have formed in the medium’s body may be determined visually.

CFU/mL (colony forming units per milliliter) is the unit of measure, and it refers to the amount of bacteria present in the original sample.

Spread plates made from a serial dilution of a liquid culture and pour plates are two examples of viable cell counts that may be performed.

Afterwards, the colonies on the plate may be counted, and the amount of bacteria present in the initial culture can be determined.

In the pour plate method, a diluted bacterial sample is combined with melted agar and then poured into a petri dish, resulting in a culture of bacteria. The colonies would be counted once more, and the number of viable cells would be determined.

Measurements of Microbial Mass

Non-target bacteria can be inhibited from growing on selective medium such as Oxoid Brilliance UTI Agar plate, which was employed in this experiment. Approximately 1uL of urine was smeared across the surface of the agar medium. One of the top samples is from a patient who has been diagnosed with clinical urinary tract infection (UTI). An example of a mixed culture is the sample at the bottom of the page. There are several methods for counting the number of bacteria in a sample. In order to determine the number of cells that are actively growing and dividing in a sample, one must first determine how many viable cells there are.

  • After a while, the colony may be seen without a microscope, and the number of colonies on a plate can be tallied.
  • It is statistically unsound to interpret less than 30 colonies, while counting more than 300 colonies frequently leads in the counting of overlapping colonies and imprecision.
  • A typical laboratory approach is diluting the sample in sterile water in successive dilutions (1:10, 1:100, 1:1000, etc.) and growing the resulting cultures on nutrient agar in a plate that is sealed and incubated for many hours.
  • coli and S.
  • coli.
  • The composition of the nutrient often contains chemicals that inhibit the development of non-target organisms while also allowing the target organism to be easily detected, which is generally accomplished by a change in the color of the media.
  • It is not necessary to use a microscope to count the colonies because the colonies are typically only a few millimeters across.

The pour plate method is used when looking for bacterial species that do not grow well in water.

Agar surfaces with sloping surfaces are created by placing the bottles on their sides and sealing them.

It is referred to as the Total Viable Count (TVC) when there are a total number of colonies (TVC).

Using a multiple of the counted number of colonies multiplied by the dilute utilized, this may be computed as follows: Spread plates made from a serial dilution of a liquid culture and pour plates are two examples of viable cell counts that may be obtained.

Afterwards, the colonies on the plate may be counted, and the amount of bacteria in the initial culture can be computed from that.

To test for bacteria using the pour plate method, dilute samples of the bacteria are mixed together with melted agar and then the mixture is poured onto a small petri dish. The colonies would be counted once more, and the number of viable cells would be determined.

Learning Objectives

Recall the many methods of determining microbial mass.

Key Takeaways

  • By determining the dry weight of a sample, it is possible to determine the cell count
  • However, the sensitivity is restricted to samples containing more than 10E8 bacteria per milliliter. In spectrophotometry, variations in turbidity are measured in order to calculate cell concentrations in an indirect manner. By counting bacteria using the plating method, which is based on the number of colonies that develop on Petri plates containing a specified growth medium, bacteria may also be counted.

Key Terms

  • Transmittance and reflectance of solutions are usually measured using a spectrophotometer, which is a kind of spectrometer. They may, however, be configured to measure diffusivity in any of the specified light ranges, which typically encompass the ranges of 200nm to 2500nm, utilizing a variety of controls and calibrations to do this. The equipment must be calibrated within specific wavelength ranges of light, and the standards used must be of a different kind depending on the wavelength of the photometric determination being made. Cell sorting and classification utilizing fluorescent markers on their surfaces is accomplished through the use of flow cytometry. A gravimeter is an instrument that measures fluctuations in the gravitational field on a local scale.

Measurement in Three Phases of Growth

During the course of bacterial growth, there are three stages: the lag stage, the log stage, and the stationary phase. Traditional microbiology training included the measurement of an exponential bacterial growth curve in a batch culture. The basic means of doing so requires bacterial enumeration (cell counting) by direct and individual (microscopic, flow cytometry), direct and bulk (biomass), indirect and individual (colony counting), or indirect and bulk (most likely number, turbidity, nutrient uptake) methods.


When it comes to determining cell mass, there are numerous options available, including thegravimetermethod, which use conventional balances to weigh a sample (dry weight/ml) after the water has been removed. Spectrophotometer: This spectrophotometer is capable of measuring samples as small as one microliter in volume. Turbidimetry is a method of estimating cell mass that is not directly comparable to other methods. Cell cultures are turbid, which means that they absorb some light while allowing the remainder to flow through.

  1. Spectrophotometers are electrical devices that are capable of measuring turbidity with extreme precision.
  2. Simple mathematical calculations are used to translate the measured turbidity to the concentration of cells in the sample.
  3. The distinction in spelling is important to note.
  4. The use of spectrophotometry does not necessitate the diluting of cultures, albeit findings become less reliable over a certain cell density.
  5. Spectrophotometry has emerged as the method of choice for fast assessments of bacterial growth and associated applications as a result of this.
  6. Additional spectrophotometers require extremely tiny amounts of culture, perhaps as low as 1 microliter of the sample in question.
  7. Additionally, the number of cells in a culture can be determined by plating the cells on the bottom of a petri dish, which is an additional method for determining microbial mass.
  8. The colonies can then be counted, and the cell concentration can be calculated based on the known volume of culture that was spread on the plate and the known number of colonies.

Furthermore, plating is the most time-consuming of all the methods: most microorganisms require at least 12 hours to form visible colonies.

Detecting Acid and Gas Production

Through the detection of acid or gas generation in culture medium, it is possible to identify between various types of bacteria.

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Learning Objectives

Display the process through which microbial acid and gas generation is measured.

Key Takeaways

  • When a microbe grows in a certain environment with appropriate nutrients or indications, it develops biochemical features that are used to distinguish it from other organisms. The presence of a pH indicator in the medium can be used to assess acid generation. It is necessary to employ the Durham tube method in order to identify gas generation by bacteria.

Key Terms

  • When a microorganism grows in a specific environment with certain nutrients or indicators, it develops biochemical properties that are used in differential media. A pH indicator can be used in the medium to determine acid generation. In order to detect gas generation by microorganisms, the Durham tube method is employed.

Cultures and Differential Media

Microbial cultures are formed utilizing a process for multiplying microbiological organisms by allowing them to grow in specified culture media under controlled laboratory circumstances, which is also known as microbiological culture or microbial culture. Microbial cultures are used to determine the type of organism being tested, as well as the amount of that organism present in the material being tested. It is one of the most important diagnostic procedures in microbiology, and it is used as a tool to pinpoint the source of infectious illnesses by allowing the agent to proliferate in a specific medium.

A “culture” is a term that can apply to the method of cultivating organisms as well as the medium in which they are produced.

When two microorganism types coexist on the same medium, differential media, also known as indicator media, can be used to differentiate one from the other.

A number of other indicators or nutrients can be used, including but not limited to: neutral red, phenol red, eosin y, and methylene blue.

Durham Cultures

When bacteria produce gas, the Durham tube technique is employed to detect this generation. Basically, they are just tiny test tubes that are placed upside down in another test tube. It is necessary to fill this little tube with the fluid in which the microbe is to be cultivated at the beginning of the experiment. If gas is formed after the inoculation and incubation, a visible gas bubble will be trapped inside the tiny tube after the inoculation and incubation. In most cases, after sterilization, which is typically conducted at 121°C for 15 minutes or so, the air gap created when the tube is put upside down is removed completely.

Escherichia coli

It can be distinguished from the majority of other coliforms by its ability to ferment lactose at 44°C when tested in the fecal coliform test, as well as by its growth and color reaction on specific types of culture media. Escherichia coli (E. coli) is a rod-shaped bacteria that belongs to the coliform group. A positive result for E. coliis metallic green colonies on a dark purple substrate was obtained when the bacteria was cultivated on an EMB (eosin methylene blue) plate.

E. coli, in contrast to the broader coliform group, are nearly exclusively of fecal origin, and their presence is therefore a reliable indicator of fecal contaminant contamination. Some types of E. coli are capable of causing significant sickness in people.

Sorbitol MacConkey Agar

SORBITOL MacConkey Agar (Sorbitol MacConkey) is a modified version of the classic MacConkey agar that is frequently employed in the detection of Escherichia coli O157:H7. MacConkey agar has traditionally been used to discriminate between bacteria that digest lactose and those that do not. Acid production may be detected by using differential media: Non harmful commensal bacteria from feces that ferment sorbitol and thrive on Cefixime Tellurite Sorbitol MacConkey Agar were isolated and cultured.

  1. Gut bacteria, such as Escherichia coli, are normally capable of fermenting lactose; however, major gut pathogens, such as Salmonella enterica and most Shigellas, are not.
  2. Acid is generated during the fermentation of sugar, resulting in a decrease in pH of the medium, which causes a change in the color of the pH indicator.
  3. In the presence of peptone, bacteria that are unable to ferment lactose, often known as nonlactose fermenters (NLFs), metabolize the sugar present in the medium.
  4. In spite of the fact that some writers refer to NLFs as colorless, in actuality, they change neutral red to a buffish tint.
  5. coliO157:H7 to ferment sorbitol, this strain differs from most other E.
  6. Lactose is substituted with sorbitol in sorbitol MacConkey agar, which is a kind of agar.
  7. coli strains digest sorbitol to generate acid: Because E.
  8. When the pH of the medium is raised, the O157:H7 strain may be distinguished from other E.


What Exactly Are Bacteria? Infections caused by bacteria, often known as germs, are minute creatures that are not visible to the human eye. Bacteria may be found everywhere, both within and outside of the body, even on your skin. Bacteria can survive in a number of settings, ranging from boiling water to ice and everything in between. Some bacteria are beneficial to your health, while others are harmful and can make you sick. Bacteria are single-celled creatures, sometimes known as simple organisms.

  1. Bacteria have a strong protective covering that increases their resistance to attack by white blood cells in the body, allowing them to survive.
  2. The flagellum is a structure that allows bacteria to move around.
  3. Bacteria may be found in abundance in the human body, particularly in the stomach and mouth.
  4. Bacteria can be classified as either aerobic or anaerobic, or as facultative anaerobes.
  5. Aerobic bacteria require oxygen in order to survive.
  6. Facultative anaerobes perform best when exposed to oxygen, although they do not require it.
  7. These bacteria aid in the digestion of meals and the preservation of your health.
  8. Bacteria are employed in the food industry to produce yogurt and fermented dishes, among other things.
  9. Examples of this include microbes that decompose dead stuff in the environment, such as dead leaves, releasing carbon dioxide and nutrients as a result of the process.
  10. Despite the fact that there are many more beneficial bacteria than dangerous bacteria, certain bacteria are detrimental.
  11. These bacteria will then emit toxins that will damage your body’s tissues and make you feel sick.

Pathogenic bacteria are bacteria that cause disease and sickness, such as strep throat, staph infections, cholera, TB, and food poisoning. They are classified as harmful since they cause disease and illness.

Rohde – Bio

Rather than referring to a rise in the size of an individual cell, the phrase “microbial growth” refers to the expansion of a population (or an increase in the number of cells). Cell division results in an increase in the number of cells in a population. Microbes are capable of two types of asexual reproduction: Fission is a basic kind of cell division that does not need the use of a spindle fiber system, and it is the mode of reproduction through which bacteria reproduce. [Most eukaryotic cells include a spindle fiber system consisting of protein filaments that is responsible for moving the chromosomes around during cell division (mitosismeiosis).

  • The bacterial cell grows in size by a factor of two and repeats its chromosomes.
  • A few bacteria and certain eukaryotes (including yeasts) have the ability to replicate via budding, which results in a bubble-like growth that grows in size and eventually splits from the parent cell.
  • Development Stages – A microbial lab culture normally progresses through four distinct, sequential growth phases that combine to produce the classic bacterial growth curve.
  • 1.Lag Phase – During the lag phase, there is no rise in the number of cells.
  • When the cells used to inoculate a fresh culture are in the log phase, this phase may not occur (as long as the inoculating parameters are the same).
  • The number of microorganisms in an exponentially rising population starts slowly at initially, but subsequently climbs tremendously quickly as the population grows exponentially.

While the number of cells does not increase, changes in the cells do occur: cells become smaller and synthesize components that allow them to survive longer periods without growing (some may even produce endospores); the signal to enter this phase may be caused by overcrowding; the signal to enter this phase may be caused by overcrowding (accumulation of metabolic byproducts, depletion of nutrients, etc.).

Death Phase – Cells begin to die during this phase of the cell’s life. Death happens at an exponentially increasing pace, although at a low rate. When a cell dies, it is because its internal ATP supplies have been depleted. Not all cells will certainly perish during this period, though.

B.Continuous Culture of Microbes

In the laboratory, cultures often go through all of the development phases; however, this is not the case in nature. Nutrients enter the cell’s surroundings at low quantities on a constant basis in nature, and populations expand at a slow but steady rate throughout the course of the year. The concentration of the scarcest or limiting ingredient determines the pace of development, not the accumulation of metabolic byproducts, because in nature there is always another microorganism that may utilise these metabolic byproducts for its own metabolism, hence restricting the rate of growth.

II.Measuring Numbers of Microbes

The majority of cultures in the lab go through all of the development phases, although this is not the case in the natural world. Nutrients enter the cell’s surroundings at low quantities on a constant basis in nature, and populations develop at a slow but steady rate throughout the course of the day. The concentration of the scarcest or limiting ingredient determines the growth rate, not the accumulation of metabolic byproducts, because in nature there is always some other microorganism that may utilise these metabolic byproducts for their own metabolism, which is why the growth rate is limited.

A. Types of Media

1.Synthetic medium – a medium created in the laboratory from components with a precise or relatively well-defined chemical makeup. 2.Complex media – a medium that contains certain components that are relatively well-known, but whose chemical composition changes significantly from batch to batch (for example, beef extracts, yeast extracts, and blood extracts); for example, nutrient agar and nutrient broth.

B. SelectiveDifferential Media(we will learn about these in detail in lab!)

A selective growth promoter is one that promotes the development of specific bacteria while suppressing the growth of others. 2.Differential – contains a component that, when a certain biological reaction happens, creates an observable change in the medium to be observed (ex. a color or pH change).

C. Controlling Oxygen Content of Media

2.Candelabras – the inoculated tube or plate is placed in a jar; a candle is lit before the jar is sealed; the burning candle consumes all of the oxygen in the jar and adds carbon dioxide to it; when the carbon dioxide extinguishes the flame, the conditions are optimal for the growth of microorganisms that require only small amounts of carbon dioxide (ex.Neisseria gonorrhoeae) 2.Thioglycollate medium – a medium that contains an oxygen-binding agent that prevents oxygen from having deleterious effects on anaerobes; the medium is often distributed in screw-cap tubes to avoid contamination.

3.Anaerobic Chamber (Brewer Jar) – A catalyst is put to a reservoir in the lid of the jar, which serves as a reservoir for the catalyst.

Hydrogen gas and carbon dioxide are produced by the breakdown of water.

A methylene blue test strip is provided in the jar to check that anaerobic conditions have been attained before proceeding.

When the strip is oxidized (when oxygen is present), it is blue; when it is reduced (when there is no oxygen), it is transparent. Return to the index of H 2 O-CO 2+ H 2H 2 +O 2-H 2 OReturn to the index of Chp.

Microbiology Introduction

  • Preparing Bacterial Cultures
  • Physical and Environmental Factors
  • Taxonomy and Identification of Microorganisms
  • Monitoring Microbial Growth
  • Preserving Bacterial Cultures
  • References. Literature

The repeatable growth of their microbial cultures is the first and most important goal of all microbiologists, regardless of whether the microbes are of natural origin or have been genetically modified by human beings. Environmental conditions that are characterized in terms of energy supply, temperature, pH, and nutrients are necessary for reproducible growth to occur (Microbial Growth Requirements). In keeping with this philosophy, we provide a variety of goods and services (see the list of culture collections, the comparison of media, and so on) that are tailored to fulfill the needs of both general microbiologists and experts.


There are simple unicellular forms (cocci, bacilli, virio, and spirillae) and multicellular forms (bacteria, fungi, and spores) in the category of creatures categorized as microorganisms (filaments and sheaths). The cyanobacteria (blue-green algae), fungi, protozoans, and bacteria are all members of this classification. Microorganisms require a source of energy and sustenance in order to live and thrive in the environment. Bacteria are the most primitive forms of microorganisms, but they are composed of a large variety of simple and complex molecules and are capable of performing a wide range of chemical transformations.

Individuals are divided into distinct nutritional categories based on their dietary requirements and the source of energy that they rely on.

Microbial growth requirements

Suitable environmental conditions, a source of energy, and sustenance are all required for microbial development to take place. Physical needs and chemical requirements are the two kinds that may be distinguished.

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Chemical factors

Natural elements essential for microbial growth are listed in a table, while chemical versions are listed in a table next to the natural elements. They also serve as buffers. 1 Complexing agents, such as EDTA or citrate, can be added to the medium to aid in the solubilisation or retention of iron in the solution.

Physical / environmental factors

The majority of microorganisms thrive at the typical temperatures that are preferred by humans, higher plants, and animals. Certain bacteria, on the other hand, can thrive at extremes (severe heat or cold) where just a few larger species can live. Bacteria are classified into three categories based on the temperature range in which they like to live: When it comes to psychrophiles (cold-loving microbes), which are found mostly in deep seawater, frozen water, and arctic environments, their optimal development temperature is between 0 and 15 degrees Celsius, with a maximum growth temperature of no more than 20 degrees Celsius.

Temperatures between 25 and 40 degrees Celsius are optimal for their development.

Heat-loving microorganisms (thermophiles) are capable of growing at high temperatures, with an optimal temperature over 60 degrees Celsius.

When placed under pressure, certain organisms may develop at temperatures close to the boiling point of water and even exceeding 100 degrees Celsius. The majority of thermophiles are unable to develop at temperatures below 45 degrees Celsius.


When the pH range between 6.5 and 7.5 is restricted to a limited range approaching neutrality, most bacteria thrive most effectively. Acidophiles (acid-loving organisms) and alkalinophiles (alkaline-loving organisms) are two classifications for organisms that thrive at extreme pH levels (base-loving). Acidophiles are bacteria that thrive at pH levels lower than 4, with some bacteria remaining alive at pH values as low as 1. Alkalinophilic bacteria prefer pH values between 9 and 10, and the vast majority of them are unable to thrive in solutions with pH values at or below neutral.

In spite of the fact that common media elements such as peptones and amino acids have a moderate buffering effect, an external buffer is required in most bacteriological media in order to neutralise acidic compounds and maintain the proper pH level.

Phosphate salts are the most commonly used buffers because they buffer within the growth range of most bacteria, are non-toxic, and serve as a source of phosphorus.

Osmotic pressure

In their natural state, microbes contain roughly 80-90 percent water, and if introduced in a solution containing a greater solute concentration, they will lose water, resulting in cell shrinkage (plasmolysis). Some bacteria, on the other hand, have evolved so successfully to high salt concentrations that they are really dependent on them for survival. These bacteria are known as halophiles (salt-loving bacteria), and they may be found in salterns and salty environments such as the Dead Sea. Figure 2: A diagram of the human body.


Aerobes are microbes that utilize oxygen for energy-producing reasons; obligate aerobes are microbes that require oxygen for their metabolism to function properly. Obligee aerobes are at a distinct disadvantage because oxygen is poorly soluble in water and because a large portion of the environment lacks this essential ingredient. In many cases, aerobic bacteria have preserved the capacity to develop in the absence of oxygen; these bacteria are referred to as facultative anaerobes. Obligate anaerobes are bacteria that are unable to utilise oxygen and, in fact, may be injured by the presence of oxygen in the environment.


Microorganisms, in contrast to larger organisms, are reliant on the availability of water for their metabolism to function properly. Microorganisms have a wide range of needs in terms of the amount of water that is accessible. Water activity and relative humidity are helpful indicators to assess the accessible water content of solids and solutions when comparing their water content.

Carbon dioxide

In autotrophic metabolisms, microorganisms capture a variety of sources of energy and reducing power, which they then employ to convert CO 2 into organic molecules. If autotrophic CO 2 -fixing microorganisms are to be grown, sodium hydrogen carbonate is typically added to the culture media, and incubation is performed in a carbon dioxide-containing atmosphere in closed vessels, or alternatively, air or carbon dioxide-enriched air is circulated through the vessel. However, while some chemoautotrophs use oxygen as the ultimate electron acceptor and derive energy from the respiration of various inorganic electron donors, other microorganisms engage in anaerobic respiration, in which they use an inorganic terminal electron acceptor other than oxygen to generate energy.

The carbon dioxide level of many bacteria found in blood, tissue, and the digestive system is greater than that found in regular air, allowing them to thrive.

Photographic bacteria, also known as phototrophic bacteria, are anaerobic organisms that use light energy to carry out a series of processes that convert carbon dioxide into triose-phosphate and other cell elements.

The removal of carbon dioxide, for example, through KOH-absorption, is consequently significant since it inhibits the development of practically all microorganisms in the environment.

Microbiological culture methodsisolating microorganisms from nature

By using a number of approaches, it is possible to isolate microorganisms from their natural surroundings. It is possible to collect microbial populations directly from the environment using a sterile swab or loop, which may then be inoculated into an appropriate liquid medium or streaked onto an agar plate if they are abundant, dense, or big enough. This is especially true for medical samples in which organisms are present in huge numbers and concentrated locations, as in a blood sample. A suitable media may also be used to directly include environmental materials containing vast microbial populations, such as soil or water.

This is accomplished by filtering the samples and incubating the filters in an appropriate medium after they have been processed.

It is possible to collect environmental samples in situ using methods such as the buried slide or buried capillary methods, which involve placing microscope slides or capillary tubes coated with a suitable medium in the natural environment (soil or sediment) and retrieving them after a specified amount of time has passed.

These approaches are used when organisms are sluggish to develop or require particular circumstances, or when the environment must be disturbed to the least amount possible.

All samples can be subcultured using any of the microbiological culture methods that have been demonstrated in this section. 3rd illustration.

Taxonomy and identification of microorganisms

Taxonomy is the study and practice of categorizing humans into categories based on their characteristics. Taxonomic approaches may be divided into three categories:

Numerical taxonomy

According to the definition, this is “the classification of taxonomic units into taxa on the basis of their properties using numerical methods.” A set of biochemical and culture assays are used to investigate all the physiological characteristics of bacteria, including the diversity of organic compounds destroyed, the necessity for specific vitamins or coenzymes, staining responses, and the inhibition of growth by antibiotics.

It is possible to code the results on a computer, and the relationships between people can be represented via a dendrogram.

The commercial availability of kits including several of these assays has facilitated the identification of a variety of bacterial strains in recent years.

Chemical taxonomy

In this case, the grouping of people is accomplished by the use of a collection of characteristics that are assumed to have been acquired from a common ancestor. In the case of chemical taxonomy, bacteria are grouped together based on the chemical similarity between the structural components of the bacteria in question. Proteins are the most often utilized materials since they are molecules that have remained mostly unchanged throughout evolution. Chemotaxonomists frequently examine the main structure of enzymes, peptidogylcan, the cytoplasmic membrane and its fatty acid content, the outer membrane, and the end products of metabolism in order to determine shared ancestry.

Molecular taxonomy

This is the comparison of the genetic sequences of chromosomal DNA or ribosomal RNA to establish similarity patterns and the phylogenetic evolution of a group. Although the DNA content in purine (G, guanine; A, adenine) and pyrimidine (C, cytosine; T, thymine) bases vary from one individual to another, they remain constant within a given species. The G+C content can therefore be used to establish taxonomic relationships. Similarities between the sequences of 16S or 23S ribosomal RNA are also compared in order to study the phylogeny of a bacterial group.

Antimicrobial sensitivity testing

When determining a substance’s antimicrobial activity, the lowest concentration of the chemical required to prevent growth of a test microorganism is often used as a standard (MlC-minimum inhibitory concentration). The tests rely on the diffusion of the antibiotic through the microbiological medium in order to impede the development of the susceptible organism that is growing in or on the microbial media. The zones of inhibition are considered to be reflective of the microbe’s sensitivity to the antibiotic treatment.

Anaerobic growth

The culture of stringent anaerobic bacteria presents a unique set of challenges since these bacteria are susceptible to being destroyed by exposure to oxygen. In the presence of metabolic electrons, dissolved oxygen in the medium reacts with the electrons to create harmful free radicals and hydrogen peroxide. Obligate anaerobes are incapable of detoxifying these active forms of oxygen because they lack the necessary enzymes. Anaerobic jars are used in conjunction with gas-generating “Gas-Paks” to grow non-stringent anaerobes on solid medium.

While in the presence of a palladium catalyst, the hydrogen combines with oxygen to form water, therefore eliminating oxygen from the jar and completing the reaction.

It is possible to purchase commercially available anaerobic chambers that are completely devoid of oxygen and filled with inert gases, which are used for the cultivation of stringent (obligate) anaerobes.

Redox potential (O/R potential)

Redox potential is defined as the ratio of oxidized to reduced molecules in a media. For example, when oxygen is dissolved in a medium, the organic compounds present become more oxidized, resulting in the medium displaying a positive redox potential. As a result of the consumption of oxygen by microbial growth, the medium’s redox potential shifts from positive to negative. During the development phase of strict anaerobes, the medium’s potential must be kept at an extremely low (negative) value.

Sodium thioglycolate (HS-CH 2 COONa) and sodium dithionite are two reducing agents that are frequently utilized because they readily transfer protons to other molecules.

Figure 4.Eh and pH ranges for microbial growth (modified from Zajic 1969): the figure was created from reports in which both pH and Eh values were provided for growth behavior in order to create the figure.

Monitoring microbial growth

It is necessary to dilute the inoculum in a succession of dilution tubes before plating them out. For each plate, the number of colonies on it is counted and then corrected for dilution to determine the number of organisms in the initial inoculum.

Most Probable Number Method

In a dilution series, a statistical approach is used to determine the most likely number of bacteria present in an inoculum that has been utilized to generate the series. An MPN is calculated by making many series with varying beginning volumes of inoculum and recording the outcomes as a sequence of positives, i.e. growth in the tube, which may then be estimated from the data to provide an MPN. The number of microorganisms that would be anticipated to produce this outcome is represented by the resultant number.

Direct Microscope Observation

It is necessary to utilize specially designed microscope slides that include a shallow well of known volume and a grid engraved into the glass in order to conduct the experiment. It is necessary to fill each grid square with the bacterial suspension, and then multiply the average number of bacteria in each square by a factor in order to obtain the counts per millilitre. Separation of bacteria from non-living material in environmental samples is accomplished by the application of selective staining (with fluorescent dyes).

Turbidity (Optical Density)

It is possible to quantify the rise in turbidity of a liquid media as a result of the growth of bacteria using a spectrophotometer. Under standardized conditions, the amount of light that reaches the detector is inversely proportional to the number of bacteria in the sample. In bacterial growth, the absorbency of the sample (optical density) is dependent on the number of cells present, as well as their size and shape, and this is measured and plotted.

Providing that absorbency measures are matched with a direct count of the same culture, its protein content, or dry mass, the correlation can be utilized in the future to estimate bacterial populations or biomass based on turbidity measurements alone.

Metabolic Activity

Using the metabolic activity of the population as a proxy for bacterial populations is another indirect method of estimating bacterial numbers. In this experiment, we evaluate the quantity of a metabolic product produced and presume that it is proportional to the number of bacteria in the sample. CO2 and organic acids, for example, are examples of metabolic products. As an alternative to the reduction test, oxygen intake can be assessed with the use of a dye, such as methylene blue, which changes color from blue in the presence of oxygen to colorless in the absence of oxygen, for example.

Preserving bacterial cultures

It is possible to utilize it for short-term storage. When kept at 4 degrees Celsius, cultures streaked on agar slants or stab cultures may remain viable for several months. In order to prevent plates from drying out, they must be sealed with an appropriate sealant. Two procedures are typically used to maintain cultures for prolonged periods of time: freezing and drying.

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Deep Freezing

When a pure culture of bacteria is suspended in a liquid, it is quickly frozen (typically with liquid nitrogen) at temperatures ranging from -50 degrees Celsius to -95 degrees Celsius. Glycerol (final concentration 15-20 percent), which works as a “antifreeze,” as well as additional protein (skimmed milk powder) are required by sensitive bacteria in order to prevent them from freezing. Cultures can be thawed and utilized for up to many years after they have been frozen.


A suspension of bacteria is swiftly frozen, and the water is extracted using a high-pressure vacuum system. They may persist in this powder-like residue for several years and can be resurrected at any moment by rehydration of the culture in nutritional medium (which is available at any time). Strains of bacteria purchased from strain collections are often given in this format.

Most Probable Number – an overview

Md LatifulBari and Sabina Yeasmin will be teaching in the Reference Module in Biomedical Sciences in 2021.

The most probable number (MPN)

The most probable number technique may be traced all the way back to the beginnings of microbiology. It is, nevertheless, still commonly used in sanitary bacteriology to estimate the quantity of coliforms present in water, milk, and other foods, among other applications. Coliform bacteria are bacteria that live in the intestines of warm-blooded mammals and are discharged in their feces on a regular basis. They are Gram-negative rods belonging to the Enterobacteraceae family that digest lactose and release gas as a result of their activity.

The MPN process is a statistical approach that is based on the idea of probability distributions.

If you want to know when the experiment has reached its conclusion, you can inoculate an appropriate growth medium with several serial dilutions and watch for the development of any recognized feature (for example, acid production or turbidity) (the presence of at least one viable microorganism in the diluted sample).

  • Replicates of three, five, and ten tubes of each dilution are given in the form of statistical MPN tables.
  • For example, the three-tube MPN approach illustrated in Fig.
  • Following the preparation of decimal dilutions of the material and inoculation of 1mL of each dilution into three broth culture tubes, the results are analyzed (different numbers of replicates and dilution series can be used).
  • The findings will be recorded as 333 if, for example, growth is observed in all of the tubes.

In accordance with APHA/AWWA/WEF 2005, the enumeration of coliforms in water was carried out using the Most Probable Number (MPN) technique (Hunt and Rice, 2005). Read the entire chapter. | Clostridium tyrobutyricum (Clostridium tyrobutyricum)

R.A. Ivy and M.Wiedmann, in the second edition of the Encyclopedia of Food Microbiology (Second Edition), 2014.


The MPN approach, which employs three or five tubes, is now the most widely used method for estimating C. tyrobutyricumnumbers in milk at the moment. The MPN of C. tyrobutyricum is often determined by adding small amounts of milk sample to a suitable medium in amounts of 0.1 ml, 1.0 ml, or 10 ml. The precise application and goal of each test determine the sample quantities (i.e., dilutions) to be utilized, as well as the number of tubes to be used each dilution. Because the late-blowing fault can be caused by as little as one or two C.

MPN testing indicates this degree of contamination when a maximum of one positive tube is found out of three tubes that contain 10 mL of milk each tube in an MPN test.

Read the whole chapter at this link: Grid Membrane Filter Techniques M.Wendorf’s article in the second edition of the Encyclopedia of Food Microbiology was published in 2014.

Calculating MPN per Gram

If all of these parameters are satisfied, the MPN for any given number of positive squares may be determined using the following formula_MPN=NlogcwhereN is the total number of grid squares on the filter; andX is the number of positive grid squares. When doing a quantitative study, the MPN per gram must always be calculated in the following order: The score is determined by counting the number of squares that contain target colonies (positive squares). If the score has been obtained over only a section of the HGMF surface (for example, 20 percent), multiply the result by the relevant factor to obtain an approximation of the score across the complete HGMF surface area.

3.To calculate the MPN per gram of sample part that has been filtered, multiply the MPN per gram by the dilution factor of the sample portion that has been filtered.

Microorganisms that reduce the amount of oil produced in high-temperature oil reservoirs (URL) Advances in Applied Microbiology, Volume 2021, Angeliki Marietou, author.

2.1.1MPN and enrichments for SRM characterization in high temperature reservoirs

Using MPN analysis, it was discovered that less than 10 SRM cells mL 1 were present in produced water samples from the Diyarbakr oil fields (Turkey), which are located between 1.6 and 2.6 kilometers below sea level and have a reservoir temperature of 55 degrees Celsius (Tüccar, Ilhan-Sungur, Abbas, and Muyzer, 2019). SRM enrichments contained a varied population of SRM connected with Firmicutes (Desporosinussp., DesulfotomaculumSp. ), Nitrospiraceae (ThermodeSulfovibriosp. ), and Proteobacteria (Desulfovibriosp.

An injection water sample and two formation water samples from oil-bearing horizons of the high temperature Samotlor (Russia) oil reservoir were enriched at 60°C with spore-forming SRM linked to the Desulfotomaculumspecies, and the enriched SRM was used to enrich the Desulfotomaculumspecies (Bonch-Osmolovskaya et al., 2003).

  • However, despite the fact that the in situ temperature reached up to 85°C, no SRM growth was recorded at temperatures more than 60°C (Bonch-Osmolovskaya et al., 2003).
  • (Okpala et al., 2017).
  • Okpala and colleagues (2017) developed a formalized formalized formalized formalized formalized formalized formalized formalized formalized formalized formalized formalized formalized formalized formalized (Okpala et al., 2017).
  • Sulfate decrease was detected at 70°C after 300 hours of incubation with rates that were comparable to or much lower than those found at lower temperatures than 55°C (Okpala et al., 2017).
  • (1993) established thermophilic enrichments using production fluids from over 30 oil producing wells, including samples from the North Sea and Alaska, to study the effects of temperature on oil output.
  • was responsible for sulfur synthesis in enrichments cultured at 85°C, which were later proven to be dominated by the bacterium (Stetter et al., 1993).
  • While incubating in a mixture of volatile fatty acids, the enrichment results were dominated by Thermodesulforhabdussp., which is capable of acetate oxidation and has an estimated doubling time of 34 hours (Kaster et al., 2007).

When lactate was used as an electron donor, Archaeoglobus sp. dominated the enrichments, despite the fact that its predicted doubling time was substantially shorter (131 h) (Kaster et al., 2007). Read the entire chapter at this link: Occurrence and Detection of Clostridium perfringens.

2016; R.Labbé and V.Juneja in the Encyclopedia of Food and Health.

Most Probable Number

When a low quantity of C. perfringens are predicted, such as in routine inspections of retail foods, an MPN method is utilized to minimize contamination. This is accomplished through the use of iron milk medium, which contains 2 percent iron powder and is both economical and simple to manufacture. The emergence of a’stormy fermentation’ is used to determine which varieties are chosen. When the milk is incubated at 45°C, casein is precipitated by the acid produced by the fermented lactose, followed by fractionation of the curd, which results in a spongy look.

Read the entire chapter.

Percival and Peter Wyn-Jones, 2014

Most Probable Number Assay

To conduct an MPNCU test, the concentrate is separated into various fractions that are then injected one at a time into a different cell culture, as shown in the diagram below. It is possible to employ dilutions of the concentrate. Each laboratory has its own protocol for culturing cells, and the number of fractions and the size of cultures might vary from five fractions each put into a 75 cm2 flask of cells to over forty fractions each inoculated into one well of a microplate. Depending on the culture, incubation might last up to 21 days.

  • When a collection of positive findings are acquired for a series of dilutions, the MPNCU is computed, where a positive culture is defined as one that exhibits characteristic CPE.
  • three or five per dilution), probability tables (Changet al.,1958) are employed, and computer programs are available for calculating the MPNCU for greater numbers of replicates (e.g.
  • This strategy is preferred by French workers, and until recently, it was also preferred by Austrian workers.
  • Ballamoole KrishnaKumar’s article in Methods in Microbiology appeared in 2018. methods of enumeration

MPN is a statistical approach that estimates the viable numbers of bacteria in a sample by inoculating broth in 10-fold dilutions. It is based on the idea of extinction dilution and is used to estimate the viable numbers of bacteria in a sample. It is often employed in the estimation of bacterial cells in both water and food. However, the approach is only capable of enumerating creatures that are still alive. It is critical to count the number of V. parahaemolyticus cells found in seafood since the Food and Drug Administration (FDA) guidelines indicate that the maximum permitted limit in seafood is 10,000 cells per gram.

  1. The three-tube MPN technique is the recognized enumeration method forV.
  2. This procedure, on the other hand, is time-consuming and labor-intensive.
  3. parahaemolyticus, it is also necessary to employ selective medium such as TCBS.
  4. parahaemolyticus, on the other hand, may be difficult to distinguish from other Vibrio and other Gram-negative bacteria that are closely linked with it.
  5. parahaemolyticus counts in seafood.

The sensitivity and reliability of molecular approaches, such as probe-based colony hybridization, are significantly higher. Read the entire chapter. Viable counts | Most Probable Number is the URL for this page (MPN)

S. Chandrapati and M.G. Williams, in the second edition of the Encyclopedia of Food Microbiology (Second Edition), published 2014.

User Interpretation of Results

When the MPN findings have been incubated for an appropriate amount of time, they are interpreted. Here, each piece of the original sample is checked for visible bacterial growth or metabolic by-products, depending on the detection method that was used in the procedure, and any visible growth or metabolic by-products are removed from the sample. Bacterial growth is demonstrated by increasing the overall sample volume, which is proportional to the number of bacteria per milliliter present in the original samples With Thomas’s approximation, the MPN in the sample may be approximated as follows: In this equation, MPNg1=P/(NT)1/2, where P is the number of positive subsamples (e.g., tubes or microwells), N is the quantity of the inocula (g) in the negative tubes, and T is the total amount of inocula (g) in all the tubes.

The results are denoted by the abbreviation MPNg1.

Read the entire chapter.

Escherichia coli is the URL for this page.

Presumptive Coliforms/ E. coli

A quantitative test for coliforms that use a most probable number (MPN) method has a variety of variables, including the kind of medium used and the temperature at which the test is carried out, among others. Using LST as an example, inoculations of a series of serial dilutions are made and the mixture is incubated at 35 °C for 24 and 48 hours, respectively. Monitoring gas production is done with an inverted Durham tube, and the tubes that are positive for gas are utilized to compute the MPN of the sample under consideration.

  1. Coliforms can be discovered by direct-pour plating with violet red bile agar (VRBA), which results in the formation of red colonies on the plate.
  2. As an alternative, coliforms can be examined on VRBA or MacConkey agar, with the pink-red colonies being selected for further testing and analysis.
  3. colitype I, but only the fecal coliforms and E.
  4. Production of indole, which can be detected using Kovac’s reagent, is characteristic of E.
  5. VRBA pour plates are an alternative to Petrifilm (a product of 3M, St.
  6. The plastic film is hydrated with water first, and then the diluted sample is placed to the plastic film surface.

The positive colonies turn red after incubation at 32 degrees Celsius. Other items have chromogenic substrates that are used to screen for the presence of glucuronidase activity. Read the entire chapter at this link: Methods for Food Hygiene Inspection

M.L. Bari and S.Kawasaki, in the second edition of the Encyclopedia of Food Microbiology (Encyclopedia of Food Microbiology, 2014).

TEMPO ®(Bio-Mérieux)

The TEMPO ®test is an automated MPN enumeration method that comprises of a vial of culture medium and a card that are particular to the test. The vial of culture medium and the card are not interchangeable. The inoculation of the cards and the reading of the cards are supported by specialized equipment and software. The sample to be analyzed is put into the vial of culture medium once it has been prepared from the main suspension. The infected medium is transferred into the card using the TEMPO ®Filler, which has three sets of 16 wells (small, medium, and large wells), with a ten-fold variation in volume between each set of wells (small, medium, and big wells).

Afterwards, the card is hermetically sealed to ensure that there is no risk of contamination during subsequent handling and storage.

It is possible to enumerate values between 10 and 49000cfuml 1, or between 100 and 490000cfuml 1, depending on the protocol.

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