Microorganisms morphology and methods of its study

Purpose of work:

to familiarize with technique and methods of microorganisms investigation.

Materials and equipment:

Microorganisms cultures, test tubes, flasks, Petri dishes, bacteriological needles, loops, streaking rods, supports,slides and cover glasses, staining agents.

Progress of work:

Test tube with culture hold in the left hand almost in horizontal position near to a torch. Before a capture of culture the right hand take out a wadded stopper from a test tube, clamping it between a little finger and a palm, and test tube edges burn on a torch flame. A needle hold in the right hand big, index and average fingers. The bacteriological needle burnt in a flame from a test tube take a small amount of microbic weight.

If culture take from the liquid environment, it is not necessary to incline strongly a test tube not to moisten its edges and a stopper. For a capture of culture it is better to use a loop. After a capture of culture of edge of a test tube and a stopper burn in a flame and close a test tube.

Hanging Drop Procedure:

1. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick. If too much Vaseline is used, it will be squeezed toward the center and mix with the drop or squeeze out the edges and get on the objective lens of the microscope.

2. Place a loopful of the culture to be tested in the center of the prepared coverslip.

3. Turn the clean concavity slide upside down (concavity down) over the drop on the coverslip so that the Vaseline seals the coverslip to the slide around the concavity.

4. Turn the slide over so the coverslip is on top and the drop can be observed banging from the coverslip over the concavity.

5. Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives.

6. Turn the objective to its lowest position using the coarse adjustment and CLOSE THE DIAPHRAGM.

7. Look through the eyepiece and raise the objective slowly using the coarse adjustment knob until the edge of the drop is observed as an irregular line crossing the field.

8. Move the slide to make that line (the edge of the drop) pass through the center of the field.

9. Without raising or lowering the tube, swing the high dry objective into position (Be sure the high dry objective is clean).

10. Observe the slide through the eyepiece and adjust the fine adjustment until the edge of the drop can be seen as a thick, usually dark line.

11. Focus the edge of the drop carefully and look at each side of that line for very small objects that are the bacteria. The cells will look either like dark or slightly greenish, very small rods or spheres. Remember the high dry objective magnifies a little less than half as much as the oil immersion objective.

12. Adjust the light using the diaphragm lever to maximize the visibility of the cells.

13. Observe the cells noting their morphology and grouping and determine whether true motility can be observed.

14. Brownian movement should be visible on slides of all the organisms, but two should also show true motility.

15. Wash the depression slide and after soaking in lysol buckets.

NOTE: The bacteria are still alive in a hanging drop slide. Slides made from possible pathogens should be soaked in lysol for 5-10 minutes with the coverslip pulled aside to expose the drop before they are washed.

Bacteria have been defined as minute vegetable cells. Their claim to a vegetable origin is based upon the experimental fact that they have the power of obtaining their nitrogen from ammonia, a property which is not possessed by animals. In form these organisms vary considerably: the most primitive is to be found in the extremely minute round or oval cells which are described as cocci (fig. 74),the variation in size being indicated by the prefix mega for the larger and micro for the smaller cocci, hence the terms megacocci and micrococci. When two circular or oval cells are joined together they are described as diplococci. When a number of cells are united to form a chain, the organism is termed streptococcus. When there is a combination of four cocci the term tetrad, or merismopedia, is applied. When the packet consists of eight divisions a sarcinacoccus is formed. When irregular heaps like bunches of grapes are found, the mass is called staphylococcus, and if irregular masses of cocci are found imbedded in a gelatinous matrix it is called ascococcus. Some micro-organisms present a rod-like character, varying considerably in length; the very short rods with rounded ends are described as bacteria, the longer ones as bacilli, which term is always used when the length of the rod is more than twice its diameter. There are also other forms, distinguished by the terms vibrios and spirilla (fig. 75), and other filamentous forms.

Cells are the fundamental units of living things, from the smallest bacterium to the largest of the plants and animals. Bacteria, the smallest cells, are visible only with the aid of a microscope. The smallest bacteria (Chlamydia and Rickettsia) are only 0.1 to 0.2 um in diameter, whereas larger bacteria may be many microns in length. A newly described species is hundreds of times larger than the average bacterial cell and is visible to the naked eye. Most species, however, are approximately 1 um in diameter and are therefore visible using the light microscope, which has a resolution of 0.2 um. In comparison, animal and plant cells are much larger, ranging from 7 um (the diameter of a red blood cell) to several feet (the length of certain nerve cells).

Each cell contains the genetic basis for reproduction in its DNA genome, the biochemical machinery for transcribing genetic information into messenger RNA (mRNA) and translating the mRNA into proteins, and the machinery for energy production and biosynthesis, which is all packaged by a membrane. In addition, each cell replicates by cell division. The mechanisms and machinery for accomplishing these functions are basically similar, but the specifics may be different for bacteria and for the higher order organisms. These differences are influenced by the structure of the cell, the environment in which the cell lives, the source and means of the cell's energy production, and the nature of and requirement for cell interaction (or lack thereof).

Control questions:

1.What each cell contain?

2.Name the smallest bacteria?

3.The procedure of a handing drop?