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.

 

Diauxic Culture and Synchronous Culture

 

Diauxic culture

The diphasic response of a culture of microorganisms based on a phenotypic adaptation to the addition of a second substrate; characterized by a growth phase followed by a lag after which growth is resumed.  In batch culture of microorganisms that have inducible enzymes, there will be two growth phases, one on glucose followed by one on the less common sugar. There is a brief delay while the needed enzymes are synthesized. Such a two phase growth is known as diauxic growth.

Catabolite repression is a type of positive control of transcription, since a regulatory protein affects an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E. coli and was originally referred to as the glucose effect because it was found that glucose repressed the synthesis of certain inducible enzymes.  When bacterium was grown in limiting amounts of glucose and lactose the plot of the bacterial growth resulted in a diauxic growth curve which showed two distinct phases of active growth. During the first phase of exponential growth, the bacteria utilize glucose as a source of energy until all the glucose is exhausted. Then, after a secondary lag phase, the lactose is utilized during a second stage of exponential growth.

The Diauxic Growth Curve of E. coli grown in limiting concentrations of a mixture of glucose and lactose

During the period of glucose utilization, lactose is not utilized because the cells are unable to transport and cleave the disaccharide lactose. Glucose is always metabolized first in preference to other sugars. Only after glucose is completely utilized is lactose degraded. The lactose operon is repressed even though lactose (the inducer) is present. The ecological rationale is that glucose is a better source of energy than lactose since its utilization requires two less enzymes.

Only after glucose is exhausted, the enzymes for lactose utilization synthesized. The secondary lag during diauxic growth represents the time required for the complete induction of the lac operon and synthesis of the enzymes necessary for lactose utilization (lactose permease and beta-galactosidase). Only then does bacterial growth occur at the expense of lactose. Since the availability of glucose represses the enzymes for lactose utilization, this type of repression became known as catabolite repression or the glucose effect.

In the presence of glucose, adenylate cyclase (AC) activity is blocked. AC is required to synthesize cAMP from ATP.  So Glucose inhibits the synthesis of cyclic AMP (cAMP), which is required for the initiation of transcription of the lac operon. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) and stimulates the binding of RNAp polymerase to the promoter for the initiation of transcription.

Thus, to efficiently promote transcription of the lac operon, lactose must be present to inactivate the lac repressor and cAMP must be available to bind to CAP that further bind to DNA and facilitate transcription. 

In the presence of glucose, adenylate cyclase (AC) activity is blocked. AC is required to synthesize cAMP from ATP. Therefore, if cAMP levels are low, CAP is inactive and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose).

Synchronous or synchronized culture

Synchronous or synchronized culture is a microbiological culture that contains cells that are all in the same growth stage. Non-synchronous cultures have cells in all stages of the cell cycle. Obtaining a culture with a unified cell-cycle stage is very useful for biological research. Since cells are too small for certain research techniques, a synchronous culture can be treated as a single cell.  Synchronous cultures have been extensively used to address questions regarding cell cycle and growth, and the effects of various factors on these.

Synchronous cultures can be obtained in several ways:

External conditions can be changed, so as to arrest growth of all cells in the culture, and then changed again to resume growth. The newly growing cells are now all starting to grow at the same stage, and they are synchronized.

Providing Limiting conditions for some time

For example, for photosynthetic cells light can be eliminated for several hours and then re-introduced. Another method is starvation of cells by eliminating an essential nutrient such as phosphate from the growth medium and later to re-introduce it. One the limiting factor is re-introduced, all the cells will start growing together and will grow in same phase.

Adding growth inhibitors for some time

Cell growth can also be arrested using chemical growth inhibitors. After growth has completely stopped for all cells, the inhibitor can be removed from the culture and the cells then begin to grow synchronously. Nocodazole, for example, is often used in biological research for this purpose.  This method has the disadvantage that the chemical molecule has to be completely removed to initiate synchronous growth.

Size dependent sorting to obtain inoculum

Cells in different growth stages have different physical properties. Cells in a culture can thus be physically separated based on their density or size. This can be achieved using centrifugation (for density) or filtration (for size).

Population of cells is fractio­nated on the basis of size. The cells are filtered so that smallest cells pass through the filter. These small cells are the youngest, and must go through their whole life cycle before dividing. Alternatively, the largest cells, which are ready to divide, may be retained or retarded by a filter. These are then collected separately and used to obtain a synchronous culture.

In the Helmstetter-Cummings technique, a bacterial culture is filtered through a membrane. Most bacteria pass through, but some remain bound to the membrane. Fresh medium is then applied to the membrane and the bound bacteria start to grow. Newborn bacteria that detach from the membrane are now all at the same stage of growth; they are collected in a flask that now harbors a synchronous culture.

The most widely used method for obtaining synchronous cultures is the Helmstetter-Cummings technique. A population of cells is passed through a membrane filter of pore size small enough to trap bacteria in the filter. The filter is then inverted, and fresh nutrient medium-is allowed to flow through it.  Loosely associated bacteria are washed from the filter.  Most bacteria pass through, but some remain bound to the membrane. Fresh medium is then applied to the membrane and the bound bacteria start to grow. Newborn bacteria that detach from the membrane are at the same stage of growth and will divide synchronously.   The method has one disadvantage, that the population size will be very small.

Helmstetter - Cummings filter pad technique

Instead of filtra­tion, density gradient centrifugation is also used to separate the cells. A population of unsynchronised cells is separated into fractions, each composed of the cells of the same density and at the same stage in their life cycle.