Quantitative Measurement of Bacterial Growth (Total & Viable Count)

In addition to knowing what types of bacteria are present in a sample, it is also important to know how many of those bacteria are there. Food manufacturers must monitor the types and numbers of bacteria that they contain for compliance with FDA requirements. After pasteurization, dairy companies monitor the number of bacteria in milk. In water treatment plants, they measure how effectively they sterilize.

Not all foods and drinks are bacteria-free. Instead, “harmless” bacteria have been greatly reduced in their numbers. As biotechnology firms manipulate bacteria to produce useful pharmaceutical products, they closely control the growth of these organisms. Distilleries monitor yeast growth during the distillation process. Antimicrobial sensitivity of a patient is determined by measuring the growth rate of bacteria in a laboratory. A number of factors can be considered to determine how many bacteria are in a sample.

For Total Population Count

Direct methods - An individual cell can be counted manually by observing each one under phase contrast microscopy on a specialized counting chamber slide. A machine, such as the Coulter Counter, can also be used to calculate the number of cells passing through a narrow passage. Milliliter counts are the result of these methods. Despite phase contrast, these techniques have some challenges, such as the fact that bacteria are small and hard to see. Bacteria often cluster together or stick together. Often, the actual number of bacteria is higher than what is reported because clumped bacteria are considered one big bacterium. Counting all bacteria present in the sample, whether dead or alive, is also part of these methods. This process takes only a short time.

Indirect methods - Turbidity may also be measured quantitatively in a spectrophotometer or qualitatively in McFarland's standard against which a total population count can be determined. Here are the steps you need to follow in order to use a spectrophotometer to measure your population. In order to standardize antimicrobial sensitivity testing, we will utilize the McFarland standards. As the number of bacteria in a sample increase, the turbidity of the sample does as well. Turbidity can be used to estimate how many viable cells are in a culture that is growing exponentially (logarithmically). In the lag phase, however, both the dead and live organisms in the culture will contribute to your total counts exceeding the viable numbers.

Viable Counts

The number of live bacteria present in a sample is often needed to determine growth rates or disinfectant effectiveness. Bacteria samples are serially diluted and then plated on appropriate growth media. Alternatively, you can place the membrane on a pad soaked in growth media and filter your samples through that. Colonies are visible when the plates have been incubated for 18-24 hours. As long as the colonies you see appear to have started from one viable bacterial unit, they are assumed to have originated from that unit. Generally, bacteria do not occur in individual form, so the colony that you see may have started from one cell or from a group of cells. CFUs (colony forming units) are used to report the results.

Plate Count Procedure

A number of methods are commonly used to count bacteria on plates, including pour plates, overlay plates, and surface counts. Through pouring and overlaying, a bacterial sample is suspended in molten agar at just the right temperature so that it cannot set up. A thin layer is then applied on top of another agar surface or poured into an empty Petri dish. Small, compact colonies are produced with these methods. Plates can be counted with a greater concentration, since the colonies won't be touching each other.

A disadvantage is the difficulty of keeping the agar hot enough to prevent it from setting up before pouring while keeping it cool enough to prevent heat shock or killing your bacteria! Surface count plates provide consistent, reliable results. Microbiology students will especially appreciate how easy it is to use! The bacteria will be pipette-applied evenly to the plate surface. Furthermore, it can be useful if you are using selective media because the bacterial growth in agar will not produce the same color response due to varying oxygen requirements. However, any bacterial culture with visible turbidity needs to be diluted down before you begin.

• As bacteria grow, they are inoculated into liquid growth mediums or solid culture media, and their populations are counted periodically.
• Multiple ways can be used to measure the growth of microbial populations. In some methods, the population is measured by its number of cells, but in others, its mass is measured, which is often directly related to its number of cells.
• Cells increase gradually in number.
• Microorganisms that reproduce by binary fission or budding also produce a large number of new cells.

Measuring the Growth of Microbes

• Various methods of measuring microbial growth can be used to determine the growth rate and generation time of microbes.
• Growth can be determined by measuring either the mass or population, which both increase as a result of growth.
• A number of different measurements can be used to measure growth:

1. In this method, the growth is measured either using an electronic particle counter or microscopy or indirectly through a colony count.
2. In this growth, the cell mass can be measured directly using an analytical weighing scale, or indirectly by measuring the nitrogen concentration in the cells or by using a spectrophotometer to determine turbidity.
3. Analyzing the degree of biochemical activity in relation to the size of the population can measure the level of cell activity in this growth.

Each measurement type will be illustrated by a specific procedure

Direct Microscopic Count

• Counting microbes directly is the most obvious method.
• Counting bacteria using the Petroff-Hausser method is the simplest and most accurate.
• The space under the cover glass and between the glass and the cover, where the bacterial suspension is held, is shown in the side view of the chamber.
• The chamber from the top. It is equipped with a grid in the center.
• This is the view of the grid enlarged. In several of the central squares, bacteria are counted at X400 to X500 magnifications.
• It is possible to calculate the cell concentration by averaging the number of bacteria in each square.
• The chamber has 25 squares each covering a part of 1 mm2. The number of bacteria in 1 mm2 is divided by the number of squares (25 squares). The number of bacteria in a cubic mm3 equals the number of squares in 25 squares (50), assuming a chamber that is 0.02 mm deep.

Electronic Enumeration of Cell Numbers

• Microbiological growth is measured by passing bacteria through tiny orifices of 10 to 30 microns in diameter within an electronic particle counter.
• The counter's two compartments are connected with an orifice containing a solution that conducts electricity.
• As bacteria pass through the orifice, the resistance between the compartments will increase momentarily. A signal is generated and automatically counted as bacterium passes through.
• The main disadvantage of this method is that it does not allow determination of whether the cells counted are viable.

Plate Count Method

• Under certain defined conditions, the number of cells that multiply in this method can be determined.
• In order to conduct a plate count, there are two different options: spreading plates or pouring plates.
• When estimating bacterial numbers, this method of bacterial counting is most commonly used in milk, water, food, and many other materials.
• A drawback to this technique is that some heat-sensitive microorganisms may be destroyed by the melted agar and thus can't colonize once it's cooled.

Turbidity Estimation of Bacterial Numbers

• When bacterial growth is being monitored, turbidity is the only practical method.
• Method for measuring bacterial growth. Microorganisms multiply in liquid media, causing the medium to become turbid with the growth of cells.
• Normally, turbidity refers to the cloudiness or haziness of a medium or fluid due to the presence of large numbers of particles.
• Spectrophotometers (or colorimeter) are instruments that measure turbidity.
• The amount of absorption of light can be used to determine microbial mass.
• A spectrophotometer transmits a beam of light through a suspension of bacteria, where a greater intensity of light is transmitted through the suspension as numbers of bacteria increase.
• The turbidity increases as the population grows due to the increase in the amount of light absorbed by the cells. Using an instrument spectrophotometer, one can measure turbidity.
• Bacterial growth can be plotted by using absorbance.

Determination of Nitrogen Content

• Cell material is mostly composed of proteins, and nitrogen is a characteristic component of proteins.
• Our ability to describe bacterial populations or cell crops can be determined by measuring bacterial nitrogen.
• Firstly, cells are harvested and washed free of medium, after which nitrogen is determined by a quantitative chemical analysis.

Determination of Dry Weight Cells

• Typical measuring methods are less reliable with filamentous bacteria and molds. A plate count cannot tell the difference between filamentous and nonfibrillar growth.
• Asexual spores are mostly counted instead of sexual ones in actinomycetes and molds.
• Growth cannot be accurately measured in this way. Dry weight is a more accurate way of determining the growth of filamentous organisms.
• It consists of removing the fungus from the growth medium, using a filter to remove extraneous materials, and drying it in a desiccator.
• It is easy to verify growth by removing a known volume of culture sample from fermentation or centrifuging it.
• It is the most convenient method for measuring the mass of cells quantitatively.

Counting Bacteria by Filtration Method

• Filtration is often used to count bacteria in lakes or streams with a relatively small number of bacteria.
• During this process 100 ml of water passes through a thin membrane filter with very small pores that prevent bacteria from passing through.
• After the filtration process, bacteria are removed from the water and remain on the filter's surface. A Petri plate containing a liquid nutrient medium is then placed on the filter surface, where colonies of bacteria grow.
• Coliform bacteria, a measure of fecal contamination of food and water, are frequently detected and enumerated using this method.

Most Probable Number (MPN) Method

• MPN (most probable number) is another method of determining bacteria numbers in a sample.
• Statistical estimating techniques rely on the principle that the greater the number of bacteria contained in a sample, the more dilution is needed in order to reduce that number to the point that no bacteria remain in the tubes after dilution.
• If the microbes on which the count is being performed are not suitable for solid media (such as chemoautotrophic nitrifying bacteria), then the MPN method is useful.
• It is also helpful for identifying microbes in liquid differential media using the growth of bacteria (such as lactose-fermenting coliform bacteria, for example).
• A MPN is just a statement that there’s a 95% chance that bacterial populations fall within a certain range and that it represents the most likely scenario.

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