MEASUREMENT OF MICROBIAL GROWTH

MEASUREMENT OF MICROBIAL GROWTH

Microbial growth to determine growth rates and generation times can be measured by different methods. Since growth leads to increase both the number and the mass of the populations, either of the two may be followed. It is necessary to make it clear that no single technique is always best; the most appropriate approach depends upon the experimental situation

Measurement of Cell Numbers

1. Breed Method

A known volume of microbial cell suspension (0.01 ml) is spread uniformly over a glass slide covering a specific area (1 sq. cm). The smear is then fixed by heating, stained and examined under oil immersion lens, and the cells are counted. Customarily, cells in a few microscopic fields are counted because it is not possible to scan the entire area of smear. The counting of total number of cells is determined by calculating the total number of microscopic fields per one square cm. area of the smear. 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 sq. mm.

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

(c) No. of cells 1 sq. cm. (or per 0.01 ml microbial cell suspension) = Average no. of microbes per microscopic field x 5000

Counting Chamber Technique

The number of cells in a population can be measured by taking direct microscopic count using Petroff-Hausser counting chamber (for prokaryotic microorganisms) or hemo-cytometers (to larger eukaryotic microorganisms). Prokaryotic microorganisms are more easily counted if they are stained or if phase I contrast of florescence microscope is employed. These are specially designed slides that have chambers of I known depth with an etched grid on the chamber bottom. , Each square on the grid has definite depth and volume. Total number of microorganisms in a sample can be calculated taking the count of number of bacteria per unit area of grid and multiplying it by a conversion factor (depending on chamber volume and sample dilution used).

1. Etched Grid	2. Cover Glass	3. Chamber Holding Bacteria

The direct microscopic method is easy, inexpensive and relatively quick to count microbial cell number. However, using these method dead cells is not distinguished from living cells and also very small cells are usually missed.

Viable Count

The bacterial culture need not contain all living cells. There might be few dead as well. Only living cells will form colony when grown in proper solid medium and under standard set or growth conditions. This fact is used to estimate number of living or dead bacterial cells (viable count) in the given culture. Estimates thus obtained are expressed as a colony f9rming unit (CFU).

Viable count technique is very much useful in the dairy industry and the food industry for quantitative analysis of milk and spoilage of food products. For convenience, to obtain a colony count for bacteria in milk, 1 ml of well mixed milk is placed in 99 ml of sterile dilute solution (may be water or nutrient broth or saline solution).

This results in s dilution of 1: 100 or 1 × 10-2. To the petri dish containing pre solidified medium 1 ml of 1: 100 dilution is transferred and incubated at desired is repeated for the preparation of further dilution as 1 : 1000 or 1 : 10, 0000 of bacteria per ml in original sample can be found by multiplying bacterial colony count by the reciprocal of he dilution and of the volume used.

For Example, CFU = 50 for 1: 10, 000 if volume used is 1 ml then,
CFU = 50 × 10, 000 × 1
CFU = 5 × 105

Coulter Counter

Coulter counter is an electronic used to count number of microbes preferably protozoa microalgae and yeasts. In This method, the sample of microbes is forced through a small orifice (small hole). On the both sides of the orifice, electrodes are present measure the electric resistance or conductivity when electric current is passed through the orifice. Every time a microorganism 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. The precaution to be taken in this method is that the suspension of samples should be free of any cell debris or other extraneous matter.

Coulter Counter

1. Electrode	2. Flow of Suspending Fluid	3. Bacterial Cell	4. Orifice
5. Electrode	6. Particle Location	7. Measurement of Voltage

Membrane-Filter Technique

Microbial cell numbers are frequently determined using special membrane filters possessing 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 separate colony. Membranes with different pore sizes are used to trap different microorganisms. Incubation times for membranes also vary with medium and the microorganism. A colony count gives the number of microorganisms in the filtered sample, and specific media can be used to select for specific microorganisms. This technique is especially useful in analyzing aquatic samples

Steps of Membrane Filter Technique

1.Membrane Filter Possessing Millipores Small Enough Trap Bacteria	Sample Filtered Through the filter to trap bacteria on the filter	3. Membrane Filter Removed and Placed in a petriplate Filled with Appropriate Medium	4. Petriplate Incubated For 24 Hours	5. Typical Colonies Develop

Measurement of Cell Mass

1. Dry Weight Technique

The cell mass of a very dense cell suspension can be determined by this technique. In this technique, the microorganisms are removed from the medium by filtration and the microorganisms on filters are washed to remove all extraneous matter, and dried in dessicator by putting in weighing bottle (previously weighted). The dried microbial content is then weighted accurately. This technique is especially useful for measuring the growth of microfungi. It is time consuming and not very sensitive. Since bacteria weigh so little, it becomes necessary to centrifuge several hundred millions of culture to find out a sufficient quantity to weigh.

Measurement of nitrogen content

As the microbes (bacteria) grow, there is an increase in the protein concentration (i.e. nitrogen concentration) in the cell. Thus, cell mass can be subjected to quantitative chemical analysis methods to determine total nitrogen that can be correlated with growth. This method is useful in determining the effect of nutrients or antimetabolites upon the protein synthesis of growing culture

Measurement of Turbidity (Turbidometry)

Rapid cell mass determination is possible using turbidometry method. Turbidometry is based on the fact that microbial cells scatter light striking them. Since the microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. One visible characteristic of growing bacterial culture is the increase in cloudiness of the medium (turbidity). When the concentration of bacteria reaches about 10 million cells (10⁷) per ml, the medium appears slightly cloudy or turbid. Further increase in concentration results in greater turbidity. When a beam of light is passed through a turbid culture, the amount of light transmitted is measured, Greater the turbidity, lesser would be the transmission of light through medium. Thus, light will be transmitted in inverse proportion to the number of bacteria. Turbidity can be measured using instruments like spectrophotometer and nephlometer

Determination of cell mass using turbidimetry method

1. Source of Light of a single wave-length (monochromatic)		2. Filter
3. Tube with cell Free medium	4. Tube with suspension of microorganisms		5. Photocell or Detector

McFarland standards

In microbiology, McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range.

Original McFarland standards were made by mixing specified amounts of barium chloride and sulfuric acid together. Mixing the two compounds forms a barium sulfate precipitate, which causes turbidity in the solution. A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl₂•2H₂O), with 9.95 mL of 1% sulfuric acid (H₂SO₄).

Now there are McFarland standards prepared from suspensions of latex particles, which lengthens the shelf life and stability of the suspensions.

The standard can be compared visually to a suspension of bacteria in sterile saline or nutrient broth. If the bacterial suspension is too turbid, it can be diluted with more diluent. If the suspension is not turbid enough, more bacteria can be added.

McFarland Nephelometer Standards:

McFarland Standard No.	0.5	1	2	3	4
1.0% Barium chloride (ml)	0.05	0.1	0.2	0.3	0.4
1.0% Sulfuric acid (ml)	9.95	9.9	9.8	9.7	9.6
Approx. cell density (1X10^8 CFU/mL)	1.5	3.0	6.0	9.0	12.0
% Transmittance*	74.3	55.6	35.6	26.4	21.5
Absorbance*	0.132	0.257	0.451	0.582	0.669

*at wavelength of 600 nm