Quantitative measurement of bacterial growth by direct and indirect methods

 

For unicellular organisms such as the bacteria, growth can be measured in terms of three different parameters:

1. Cell number                        2. Cell mass               3. Cell activity

Methods for Measurement of Cell Numbers

Cell numbers could be determined either by direct counting or by indirect methods.

The most obvious way to determine microbial numbers is through direct counting. Using a counting chamber is easy, inexpensive, and relatively quick; it also gives information about the size and morphology of microorganisms. It will also reveal the presence of bacteria that do not form colonies on the media used or the conditions under which it may be incubated; thermophiles, psychrophiles, and dead bacteria would fall in this category.

Petroff-Hausser counting chambers can be used for counting prokaryotes. The counting is easy if the organisms are stained, or when a phase-contrast or a fluorescence microscope is employed. These specially designed slides have chambers of known depth with an etched grid on the chamber bottom. The number of microorganisms in a sample can be calculated by taking into account the chamber’s volume and sample dilutions required. There are some disadvantages to the technique. The microbial population must be fairly large for accuracy because a small volume is sampled. It is also difficult to distinguish between living and dead cells in counting chambers without special techniques.

The bacteria in several of the central squares are counted, usually at X400 to X500 magnifications. The average number of bacteria in these squares is used to calculate the concentration of cells in the original sample. Since there are 25 squares covering an area of 1 mm², the total number of bacteria in 1 mm² of the chamber is (number/square) x (25 squares).

The chamber is 0.02 mm deep and therefore,

Bacteria/mm³ = (bacteria/square) x (25 squares) x (50).

The number of bacteria per cm³ is 10³ times this value.

For example, suppose the average count per square is 20 bacteria:

Bacteria/cm³ or Bacteria / ml = (20 bacteria) x (25 squares) x (50) x (10³) = 2.5 × 10⁷.

Coulter counter

Larger microorganisms such as protozoa, algae, and non-filamentous yeasts can be directly counted with electronic counters such as the Coulter Counter. The microbial suspension is forced through a small hole or orifice. An electrical current flow through the hole, and electrodes placed on both sides of the orifice measure its electrical resistance. Every time a microbial cell passes through the orifice, electrical resistance increases (or the conductivity drops) and the cell is counted. The Coulter Counter gives accurate results with larger cells and is extensively used in hospital laboratories to count red and white blood cells. It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems.

Counting chambers and electronic counters yield counts of all cells, whether alive or dead.  Culturing methods like plate count or membrane filter count yield viable bacterial count.

Breeds count

This is also a direct microscopic counting procedure, particularly used for quantitation of bacteria in milk.  This is accomplished by staining a measured amount of milk (0.01 ml) that has been spread over an area one square centimeter (1 cm²) on a slide. The slide is examined under microscope and the organisms in single microscopic field are counted. To increase accuracy, several fields are counted to get average field counts.  The count could be then converted to count per milliliter, if we know the area of a single field. 

The total number of cells can be counted with the help of following calculations:

(a) Area of microscopic field = πr²

r (oil immersion lens) = 0.08 mm.

Area of the microscopic field under the oil immersion lens

= πr² = 3.14 x (0.08 mm)² = 0.02 mm².

(b) Area of the smear one cm². = 100 sq. mm. Then, the no. of microscopic fields = 100 / 0.02= 5000

(c) No. of cells per one cm² (or per 0.01 ml microbial cell suspension) = Average no. of microbes per microscopic field x 5000

Standard Plate Count (Viable Count)

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. The suspension is either spread onto the surface of agar plates (spread plate method), or is mixed with molten agar, poured into plates, and allowed to solidify (pour plate method). The plates are then incubated under conditions that permit microbial reproduction so that colonies develop that can be seen without the aid of a microscope. It is assumed that each bacterial colony arises from an individual cell that has undergone cell division. Therefore, by counting the number of colonies and accounting for the dilution factor, the number of bacteria in the original sample can be determined.

The viable count is an estimate of the number of cells. Because some organisms exist as pairs or groups and because mixing and shaking of the sample does not always separate all the cells, we may get a count of "colony forming units". One cell or group of cells will produce one colony, and thus viable count is measured as colony forming units.

Since we don’t know how many bacteria may be present in a sample, we used to prepare a dilution series to ensure that we obtain a dilution containing a reasonable number of bacteria to count (approximately 30-300). Dilutions in the range 10^-1 (1/10) to 10^-8 (1/100,000,000) are generally used.

The major disadvantage is that the nature of the growth conditions, including the composition and pH of the medium used as well as the conditions such as temperature, determines which bacteria in a mixed population can grow. Many others will not be able to grow.  This technique is advantageous for quantitation of specific microbial population where we can design the conditions such that the desired organisms can grow.

Viable Count using Membrane filter

Microbial cell numbers can be determined using special membrane filters that have millipores small enough to trap bacteria. In this technique a water sample containing microbial cells passed through the filter. The filter is then placed on solid agar medium or on a pad soaked with nutrient broth (liquid medium) and incubated until each cell develops into a colony. Membranes with different pore sizes are used to trap different microorganisms. Incubation times also vary with medium and the microorganism.

Most Probable Number technique

The Most Probable Number (MPN) is widely used to estimate numbers of coliforms in water, milk, and other foods. Coliforms are bacteria that reside in the intestine of warm-blooded mammals and are regularly excreted in the feces. They are Gram negative rods belonging to the Enterobacteriaceae family, ferment lactose and produce gas.

The MPN procedure is a statistical method based upon the probability theory. Samples are serially diluted to a point where there are no more viable microorganisms. To detect the end point, multiple serial dilutions are inoculated into a suitable growth medium, and the growth is monitored. The pattern of positive tests (growth) in the replicates and statistical probability tables are used to determine the concentration (most probable number) of bacteria in the original sample. Statistical MPN tables are available for replicates of 3, 5, and 10 tubes of each dilution. The more replicate tubes used, the greater the precision of the estimate of the size of the bacterial population.

These techniques are based upon statistical probabilities with the assumption that there is a uniform distribution of bacteria in liquid or homogenized samples. Growth and multiplication in a suitable broth can be detected by manifestations such as turbidity or acid and gas production. These methods can be used for most bacteria, but they are commonly used for the detection of coliform bacteria in water supplies. MacConkey broth or lactose broth with Brilliant green as pH indicator is often used in coliform counts. Acid production is indicated by colour change of the broth and gas is trapped in a Durham tube.

By referring to standard MPN probability tables, the MPN of bacteria can be determined.

Methods for Measurement of Cell Mass

Methods for measurement of the cell mass involve both direct and indirect techniques.

1. Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation.

Dry cells weight (DCW)

The most commonly used direct method for determination of cellular dry weight. It is applicable only for cells grown in solid-free medium.  Samples of culture broth are centrifuged or filtered and washed with a buffer solution or water.  The washed wet cell mass is then dried at 80℃ for 24 hr, and dry cell weight is measured.

Packed cell volume (PCV)

It is used to rapidly but roughly estimate the cell concentration in a fermentation broth.  Fermentation broth is centrifuged in a tapered graduated tube under standard conditions and the volume of cell is measured.  It is used for indirect method for measurement of mold, fungus, and Actinomycetes with hypha.

2. Direct chemical measurement of some chemical component of the cells such as total Nitrogen, total protein, or total DNA content.

1) Measurement of DNA, RNA, ATP, NADH, and CO₂

2) Measurement of protein by Biuret method or Lowry method

Bacterial counts can be assessed by measuring bacterial ATP with the bioluminescence luciferin-luciferase technique. 

The determination of adenosine triphosphate (ATP) with a bioluminescence assay is used in the detection of viable bacteria. Here ATP (the important compound in metabolism that is found within all living cells) is quantitated. The assay is based on the reaction between the luciferase (enzyme), luciferin (substrate), and ATP. Light is emitted during the reaction, and can be measured quantitatively and correlated with the quantity of ATP and thus to the quantity of bacteria.  The total light emitted during the course of the reaction is a function of the concentration of luciferase, luciferin, oxygen and ATP. By keeping luciferin, luciferase and oxygen in excess, the maximum intensity of the emitted light will proportional to the ATP concentration.

However, when applied to the measurement of bacterial ATP in clinical samples such as urine, bioluminescence presents certain problems. Firstly, bioluminescence will detect ATP from both mammalian and microbial sources so that non-bacterial ATP must be released and destroyed before the assay. Secondly, substances present in urine can inhibit the luminescent enzyme reaction. Lumac markets a kit for the detection of bacteriuria in which bacterial ATP is assayed by luciferin-luciferase bioluminescence. Host ATP is extracted from cells and both free and extracted ATP are removed by treatment with the ATP destroying enzyme apyrase; bacterial ATP is then extracted and, after the addition of luciferin-luciferase reagent, measured by bioluminescence.

Turbidity or visocity measurements 

Turbidity measurements employ a variety of instruments to determine the amount of light scattered by a suspension of cells.  Particulate objects such as bacteria scatter light. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number. The method is simple, but the sensitivity is limited to about 10⁷ cells per ml.  The intensity of the transmitted light is measured using a spectrophotometer or calorimeter.  It provides a fast, inexpensive, and simple method of estimating cell density.

Methods for Measurement of Cell activity

Indirect measurement of chemical activity such as rate of O₂ production or consumption, CO₂ production or consumption, etc.