Determination of Nitrogen constituents,dry weiht of cells and pure culture technique

Determination of Nitrogen constituents

The major constituent of cell material is protein and since nitrogen is a characteristic part of protein, one can measure the bacterial population or cell crop in terms of bacterial nitrogen. Bacteria average approximately 14% nitrogen on a dry weight basis. Although this figure is subject to some variation introduced by changes in culture condition or differences between species. To measure growth by this technique. First of all, cells should be harvest and wash them free of medium and then perform a quantitative chemical analysis for nitrogen. Bacterial nitrogen determination is somewhat laborious and can be performed only on specimens free of all other sources of nitrogen. Furthermore,the method is applicable only for concentrated populations. For these and other reasons this procedure is used primarily in research.Growth is expressed in MgN₂/ml.

Determination of the dry weight of cells.

This is the most direct approach for quantitative measurement of a mass of cells. However, it can be used only with very dense suspensions and the cells must be washed free of all extraneous matter. However, dry weight must not be always indicative of a number of living materials in cells. For eg;the intracellular reserve material poly-[bita]-hydroxybutyrate can accumulate in Azotobacter beijerinchii at the end of the log phase of growth and during the stationary phase and finally can comprise up to 74% of the dry weight of the cells. Thus the dry weight may continue to increase without corresponding cell growth. Yet, for many organisms, the determination of dry weight is an accurate and reliable way to measure growth and is widely used in research. The growth is determined in Mg dry weight of cell per ml.

Measurement of a specific chemical change produced on a constituent of the medium

A species that produces an organic acid from glucose fermentation may take as an example of this method of estimating cell mass. The assumption is that the amount of acid produced under the specific condition and during a fixed period of time is proportional to the magnitude of the bacterial population.Admittedly the measurement of acid or any other end product is a very indirect approach to the measurement of growth and is applicable only in special substances. Growth is expressed in milliequivalents of acid per ml or per culture.

Pure culture technique

In our environment (soil,air,water,sewage,body surfaces) microorganism exists in the mixed population. It is only in a very rare situation that they occur as single species. To be able to study the cultural, morphological, physiological and immunological characteristics of a species it is essential,first of all, that it be separated from the other organisms normally found in its habitat;in another word, we must have a pure culture of the microorganism. A culture that contains only one kind of microorganism is called a pure culture. A culture which contains more than one kind of microorganism is called mixed culture. Two kinds of operation ie isolation (the separation of particular microorganism from the mixed populations that exist in nature) and cultivation (the growth of microorganism in artificial environments such as culture media under laboratory conditions) come inti play irrespective of the kind of microorganisms with which a microbiologist deals.

A pure culture of the microorganism that form discrete colonies on solid media may be most simply obtained by one of the modifications of the plating method. Each viable organism gives rise through growth to a colony from which sub-culture can be readily made. Several different methods of getting a pure culture from a mixed culture are

• Streak plate
• Pour plate and
• Spread-plate method
• In all these methods the technique involves thinning out the organism so that the individual species can be selected from the others.

Streak plate method:

The streak plate method is a most economical and practical method of obtaining discrete colonies and pure cultures.In this method, a sterilized loop is dipped into a suitably diluted suspension of organisms which is then streaked on the surface of an already solidified agar plate to make a series of parallel, non-overlapping streaks. Streaking can be made in a different way.

Procedure

1. Label all the plates, on the bottom,with the name of the sample to be inoculated with the help of a marker.
2. Hold the tube containing the sample in the left hand.
3. Sterilize the loop on the flame holding in the right hand.
4. Remove the cotton wool plug from the sample tube and immediately flame the mouth of the tube.
5. Introduce the loop into the sample tube and withdraw one loopful suspension.
6. Flame the mouth of the tube,replace the cotton wool plug and place the tube in the test tube rack.
7. Lift the Petri plate cover with the left hand and hold it at an angle of 60^.
8. Streak the plate by any one of the following methods:

• Quadrant streak method:
• Spread one loop full of organism over a small area near the edge of the plate and streak it. This is area 1,apply the loop lightly to the medium to avoid digging into it.
• Streaking the loop by flaming and cool it for 5 seconds.
• Make five or six area from area 1 through area 2. Stay near the edge of the plate.
• Sterilize the loop again and allow it to cool.Make six or seven streaks from area 2 through area 3.
• Sterilize the loop again and make as may streaks from area 3 through area 4 as possible.
• Sterile the loop again before putting it down

Radiant streak method

• Spread one loopful organism over a small area near the edge of the plate. This is area 1.
• Sterilize the loop and allow it to cool for 5 seconds.
• From the edge of area 1 make seven or eight streaks to the opposite side of the plate.
• Sterilize the loop again and cross-streak over the last group of streaks.
• Flame the loop again before putting it down.

T streak method

• With the help of marker,draw a "T" on the bottom of plate dividing the plate into one-half and two-quarters.
• Inoculate the half portion with one loopful of the organism in a continuous line from the edge of the plate to the middle of the plate.
• Sterilize the loop and cross-streak from area 1 into area 2 with one continuous streak, filling area 2.
• Sterile the loop again and cross-streak from area 2 to area 3 with a single continuous streak.

Continuous streak

• Starting at the edge of the plate with a loopful of organisms, spread the organisms in a single continuous movement to the center of the plate.
• Rotate the plate 180 so that the uninoculated portion of the plate is away from you.
• Without flaming loop, and using the same face of the loop,continue streaking the other half of the plate by starting at area B and working toward the center.
• Flame your loop before setting it aside.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.