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Direct
Microscopic
Counts

H err Gustav Druer, the Brno wine merchant has a problem and Brother Gregory has been asked to help.

You are to become his research assisitants and help him carry out a research investigation into the properties of microbes.

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A batch of new wine has become contaminated with microbes that are spoiling the wine and turning it sour. Brother Gregory has isolated the microbe and is now trying to determine its growth properties. He wants you, his research assistants, to try growing this microbe under a number of different environmental conditions and find out how fast it reproduces.

Background All organisms, from giant elephants down to microbes so small they can only be seen with a microscope, need a favorable environment in which to grow and reproduce. Nutrients are essential, and a range of chemical substances that includes organic and inorganic elements must be provided within this environment.

Some microorganisms, like the ones Brother Gregory wants you to investigate, use organic nutrients to provide themselves with energy and a source of carbon to use as building blocks for their own structures, these are the chemoorganotrophs. But in the complex world of microorganisms, other strategies are also used.

The chemolithotrophs obtain their energy from a chemical reaction between oxygen and inorganic compounds such as hydrogen sulfide (H2S), ammonia (NH3), or even ferrous iron (Fe++). Phototrophs are able to harvest sunlight and convert the radiant energy into chemical energy. They usually store this energy as carbohydrates.

All life on earth is based on the special chemical properties of carbon, so microorganisms need a constant supply of this element in a form that they can use. Heterotrophs used preformed organic materials ("food") as their source of supply, while autotrophs can use carbon dioxide.

Most microorganisms live and grow in liquid environments, and can only survive within narrow bands of critical environmental conditions. Temperature and pH are two of these conditions. Some microorganisms can tolerate, and grow at temperatures of -10oC, or above 100oC, and flourish at concentrations of hydrogen ions (pH) that would kill almost any other creature. But most grow best under less extreme conditions.

Measuring Growth
Bacteria, such as the ones you will be investigating here, reproduce by first enlarging in size, and then dividing into two separate, smaller cells. During the enlargement phase, cells synthesize all their internal cellular structures, protein, membranes, etc., including the continuous synthesis of its genetic DNA. It is during this phase that the cells must take up nutrients, catalyze reactions with enzymes, and eliminate wastes.

In the phase called binary fission, a septum, or "separator", consisting of membrane and new cell wall, is synthesized across the cell, dividing it into two equal parts. In many species, the two new daughter cells separate from each other become independent entities.

The time it takes for cells to complete the enlargement phase and the division phase is called the generation time, (or sometimes, the "doubling time"). Growth, therefore, can be measured either by determining the increase in mass of a population of cells, or by counting the increase in cell number over a period of time.

In this investigation you will be measuring the growth of a microbe population by directly counting the number of cells that are present at various times, and under a variety of conditions.

special section
Mendel's Mother shows you --- -- how bacteria grow.

Tools of the Trade
Microbe populations are easy to manipulate and easy to maintain. They grow rapidly and produce large numbers of individuals in very short periods of time (hours), so experiments are cheap, fast and consistent.

A tiny group of the microbes under investigation are placed into a liquid, nutrient filled broth that has been sterilized (so no other bacteria will compete!). Usually this is in a special flask (called an "Erlenmeyer flask"), which is slowly shaken (to keep the microbes and nutrient at uniform distributions), and at kept the appropriate constant temperature.

At regular intervals of time, small samples of the growing culture are taken from the flask and all reproduction of the microbes stopped by some poison or inhibitor (they can also be chilled or frozen). The size of the population at each time point can then be determined in several ways, but the most direct way is to count the individual microbes, using a microscope.

After shaking the sample vigorously (to make sure all the microbes are evenly suspended), a small drop of the suspension is placed on a special microscope slide called a hemocytometer, or Petroff-Hausser counting chamber.

A clear area at the center of this special slide is divided, by colored lines, into carefully calculated areas. A coverslip is then placed over the suspension on the grid, and held a very accurate distance away from the surface. The microbes are thus contained within a small, but known, volume of liquid, and can easily be counted by looking at the chamber through an appropriate microscope lens.

Most Petroff-Hausser counting chambers consist of 400 small squares each with an area of 0.0025 mm2, 25 large squares with areas of 0.04 mm2 and the suspension depth (height of the coverslip) is 0.02 mm.

An investigator places a small sample of the microbe culture between the grid (on the surface of the slide) and the coverslip and makes several counts of the number of cells found within one or more of the larger squares. These numbers are recorded. The total number of cells in a milliliter of the original growth population can then be calculated.

special section
Mendel's Mother shows you ---
-- how to use the counting chamber.

Dilutions
Microbe cultures grow rapidly and populations of billions of cells per milliliter of culture are soon reached.

Direct microscopic examination of such a well grown culture on a counting chamber is impossible because of the large numbers of cells that crowd together and fill the field of view.

Before examining and trying to count such cultures, appropriate dilutions of each sample must be made. A known volume of the original population is taken and placed into a larger volume of sterile water (or other liquid). This mixture is shaken (to make is consistent), and then a sample is taken for placement in the counting chamber and microscopic examination. The new sample is less concentrated (more dilute), so the cells are further apart and thus easier to see.

For example, if 1 ml (one milliliter) of the original culture is placed in 9 ml of water, this represents a ten fold dilution (1 to 10 dilution). The microbes in this new sample are ten times LESS concentrated than before. If the investigator counts 15 microbes in 1 milliliter of this new sample, it means that there were 15 x 10 = 150 microbes in the original culture.

The dilution factor, is the amount a sample has been diluted, and therefore the amount by which the counted number of cells must be multiplied to get to the correct concentration (or total).

Much larger dilution factors are also used. For example, if 0.1 ml of the original culture is placed into 9.9 ml of sterile water, this represents a 1 to 100 dilution, and is equivalent of placing 1 ml of the original culture into 99 ml of water. If 0.01 ml is placed into 9.99 ml of water, this would be a 1 to 1000 dilution factor.

Very large dilution factors are sometimes needed before well grown bacterial and microbe cultures can be accurately counted using the direct microscopic method.

Recording Results

CLICK HERE,
print out, and use this
Table of Results
to record your data

The results of each of your investigations should be recorded as a table (a Table of Results). In these tables you should indicate the conditions of growth, and make an accurate record of both the time intervals and the number of cells you counted at each sample point.
special section
Mendel's Mother's calculator ---
-- use it to calculate log values.

Since bacteria and other microbes grow exponentially for this part of your investigation, it is usual to convert the value of the number of cells counted into the logarithm of that number (Log. cell number). This log. value should also be recorded on your table of results.

Graphs

CLICK HERE,
print out, and use this
Presenting the Results
sheet to graph your data

The results of each investigation should then be presented as a graph.

In each case, the horizontal axis of the graph should be the time intervals at which the samples were taken.

The vertical axis should represent the value of the number of cells counted in that particular sample at that time point.

Plotting the direct cell counts (raw data) will produce a curved line that illustrates exponential growth (green line). However, the number of cells at each time point rapidly becomes very large.

A different type of graph is obtained it each count of the cell number is first converted (using the calculator, above) to the logarithmic value for that numnber. When this number is be plotted on the vertical axis, the final graph is called a semilogarithmic plot (red line).


------ begin growing cells.

 


Conditions;
Samples;
Dilutions;
Each investigation is carried out under a specific set of growth conditions.

There are three possible temperatures; cool, warm and hot, and two different amounts of nutrient in the growth broth; low and high.

Each set of different growth conditions will affect how the microbes grow and what kind of growth curve they will produce.

All investigations start at time 0 hours, when a small sample of microbes are placed in fresh, sterilized growth broth and the flask placed at the appropriate temperature. The microbes are allowed to grow for a maximum of 10 hours. Samples can be taken at every hour.

At early sampling times the growth broth will not contain many cells, so the sample can be examined in the Petroff-Hauser counting chamber directly and undiluted.

However, as the cells, grow, divide and increase in number it is necessary to dilute the sample before it can be counted under the microscope. An appropriate dilution value must be chosen before the cells in the counting chamber can be accurately counted.

It is important to record, and take into account, the amount by which a sample has been diluted before reporting the results.


Science at a Distance
© 2000, Professor John Blamire